Abstract
Scavenger receptor class B, type I (SR-BI) mediates selective uptake of high density lipoprotein (HDL) lipids. It is unclear whether this process occurs at the cell membrane or via endocytosis. Our group previously identified an alternative mRNA splicing variant of SR-BI, named SR-BII, with an entirely different, yet highly conserved cytoplasmic C terminus. In this study we aimed to compare HDL uptake by both isoforms. Whereas SR-BI was mainly ( approximately 70%) localized on the surface of transfected Chinese hamster ovary cells, as determined by biotinylation, HDL binding at 4 degrees C, and studies of enhanced green fluorescent protein-tagged SR-BI/II fusion proteins, the majority of SR-BII ( approximately 80-90%) was expressed intracellularly. The cellular distribution of SR-BI was not affected by deletion of the C terminus, which suggests that the distinct C terminus of SR-BII is responsible for its intracellular expression. Pulse-chase experiments showed that SR-BII rapidly internalized HDL protein, whereas in the case of SR-BI most HDL protein remained surface bound. Like its ligand, SR-BII was more rapidly endocytosed compared with SR-BI. Despite more rapid HDL uptake by SR-BII than SR-BI, selective cholesteryl ether uptake was significantly lower. Relative to their levels of expression at the cell surface, however, both isoforms mediated selective uptake with similar efficiency. HDL protein that was internalized by SR-BII largely co-localized with transferrin in the endosomal recycling compartment. Within the endosomal recycling compartment of SR-BII cells, there was extensive co-localization of internalized HDL lipid and protein. These results do not support a model that selective lipid uptake by SR-BI requires receptor/ligand recycling within the cell. We conclude that SR-BII may influence cellular cholesterol trafficking and homeostasis in a manner that is distinct from SR-BI.
Highlights
SR-BI is a ϳ82-kDa protein with two short cytoplasmic termini, two transmembrane domains, and a large, heavily glycosylated extracellular loop [2]
The surface expression of both receptors was unchanged by 5 h of exposure of cells to high density lipoprotein (HDL) in the medium, as opposed to cells not exposed to HDL (Fig. 1B), indicating that the presence of the ligand did not induce the redistribution of receptors
These results indicate that the internalization of HDL by SR-BII into the transferrin-containing endosomal recycling compartment (ERC) occurred at a relatively rapid rate compared with SR-BI
Summary
SR-BI is a ϳ82-kDa protein with two short cytoplasmic termini, two transmembrane domains, and a large, heavily glycosylated extracellular loop [2]. Only the extracellular loop of SR-BI is essential for selective uptake [13,14,15]; both the N- and C-terminal cytoplasmic tails can be deleted or exchanged with the corresponding region of the other class B receptor CD36 [14, 15]. In polarized hepatocytes, the interaction of the C terminus of SR-BI with a protein called CLAMP, or PDZK1, is required for cell surface expression of the receptor [17, 18]. SR-BII protein was detected in human retinal epithelial cells [22] and in rat Leydig cells [23] Another indicator for a possible biological function of SR-BII is the high degree of conservation of the C terminus between various species [19]. Our results suggest that selective lipid uptake by SR-BI does not require receptor/ligand recycling
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