SR-BII, an Isoform of the Scavenger Receptor BI Containing an Alternate Cytoplasmic Tail, Mediates Lipid Transfer between High Density Lipoprotein and Cells

Abstract

The scavenger receptor class B, type I (SR-BI), binds high density lipoprotein (HDL) and mediates selective uptake of cholesteryl ester from HDL and HDL-dependent cholesterol efflux from cells. We recently identified a new mRNA variant that differs from the previously characterized form in that the encoded C-terminal cytoplasmic domain is almost completely different. In the present study, we demonstrate that the mRNAs for mouse SR-BI and SR-BII (previously termed SR-BI.2) are the alternatively spliced products of a single gene. The translation products predicted from human, bovine, mouse, hamster, and rat cDNAs exhibit a high degree of sequence similarity within the SR-BII C-terminal domain (62-67% identity when compared with the human sequence), suggesting that this variant is biologically important. SR-BII protein represents approximately 12% of the total immunodetectable SR-BI/II protein in mouse liver. Subcellular fractionation of transfected Chinese hamster ovary cells showed that SR-BII, like SR-BI, is enriched in caveolae, indicating that the altered cytoplasmic tail does not affect targeting of the receptor. SR-BII mediated both selective cellular uptake of cholesteryl ether from HDL as well as HDL-dependent cholesterol efflux from cells, although with approximately 4-fold lower efficiency than SR-BI. In vivo studies using adenoviral vectors showed that SR-BII was relatively less efficient than SR-BI in reducing plasma HDL cholesterol. These studies show that SR-BII, an HDL receptor isoform containing a distinctly different cytoplasmic tail, mediates selective lipid transfer between HDL and cells, but with a lower efficiency than the previously characterized variant.

Serum Amyloid A Is a Ligand for Scavenger Receptor Class B Type I and Inhibits High Density Lipoprotein Binding and Selective Lipid Uptake

“…CHO cells were grown in Hams F12 medium supplemented with 5% (v/v) heat-inactivated fetal bovine serum, 2 mM L-glutamine, 50 units/ml penicillin G, and 50 g/ml streptomycin. Clones were screened for expression of the scavenger receptors by immunoblotting with a rabbit polyclonal antibody recognizing the extracellular domain of SR-BI (18). For biochemical assays, CHO cells were grown in 12-well clusters (Corning Corp., Corning, NY).…”

“…For studies involving selective lipid uptake, HDL was double-labeled by iodination of the protein component and by tracing the CE component with nonhydrolyzable, intracellularly trapped [1,2(n)-3 H] cholesterol oleyl ether as described previously (20). Briefly, [1,2( Ligand Binding and Uptake Assays-Cell association assays were performed as described previously (18,21). Briefly, cells were seeded into 12-well cell culture clusters at an initial density of 1 ϫ 10 5 cells/ cm 2 .…”

“…Cell Culture-Stable transfectants of CHO cells expressing human SR-BI (CHO-SR-BI cells) were generated as described elsewhere (18) and cultured in medium containing 0.25 g/liter geneticin (Invitrogen). CHO cells were grown in Hams F12 medium supplemented with 5% (v/v) heat-inactivated fetal bovine serum, 2 mM L-glutamine, 50 units/ml penicillin G, and 50 g/ml streptomycin.…”

Trophoblast‐like human choriocarcinoma cells serve as a suitable in vitro model for selective cholesteryl ester uptake from high density lipoproteins

“…Protein transfer to nitrocellulose membranes was performed electrophoretically at 150 mA, 4°C, 50 min [34]. Immunochemical detection of SR-BI and SR-BII (a splice variant of SR-BI lacking the C-terminal portion [29]) was performed with a sequence-specific rabbit anti-SR-BI-peptide (496-509) and anti-SR-BII peptide (491-506) IgG (dilutions 1 : 1500, Abcam, Cambridge, UK). Immunoreactive bands were visualized with peroxidase-conjugated goat anti-rabbit IgG and ECL development [30].…”

“…High level adenoviral-overexpression of SR-BI resulted in an enhanced and comparable capacity for selective CE-uptake by all three choriocarcinoma cell lines. The lack of SR-BII protein suggested that this splice variant of SR-BI [29] does not contribute to selective CE-uptake by BeWo, JAr, and Jeg3 cells.…”