Mulcahy, Jane Victoria; (2002) Human scavenger receptor class B, type II (SR-BII) and cellular cholesterol efflux. Doctoral thesis (Ph.D), UCL (University College London).

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Abstract

A key feature of atherosclerotic lesions is the presence of cholesteryl ester (CE)-engorged macrophages within the arterial intima. Cholesterol mobilisation from intracellular stores involves high-density lipoprotein (HDL) binding to an unknown cell surface receptor, activation of signalling pathways, and translocation of free cholesterol to the plasma membrane for efflux. The scavenger receptor, class B (SR-B) gene encodes for two different splice variants (types I and II), which have identical extracellular regions that bind HDL but C-terminal cytoplasmic tails that differ. I identified a number of putative signalling motifs in the C-terminus of SR-BII, which are absent from SR-BI, and hypothesised that: upon HDL stimulation of SR-BII, its C-terminal cytoplasmic tail interacts with a signalling molecule to activate the CE mobilisation pathway. Pull-down assays to screen potential binding proteins revealed that biotin-tagged cytoplasmic SR-BII interacts with the Src Homology 3 domain of phospholipase C-γ1, in an isoform-specific manner. However, I failed to detect this interaction within more physiological, cellular environments. Full-length SR-BI or SR-BII sequences were over-expressed in Chinese hamster ovary cells (CHO-SR-BI/II), and caveolae (cholesterol-rich microdomains of the plasma membrane) were isolated from cell lysates using sucrose density gradient ultracentrifugation. Human SR-BII was detected in this membrane fraction, while co-immunoprecipitation with caveolin-1 confirmed its true caveolar localisation. This finding implicates SR-BII in both signalling and cholesterol flux; two functions associated with caveolae. Following the generation of an isoform-specific human SR-BII antiserum, cell surface expression of SR-BII protein in THP-1 monocytes and macrophages was revealed. To investigate the role of this isoform in CE mobilisation, recombinant CHO-SR-BII cells were labelled with 3H-oleic acid and reductions of cellular cholesteryl oleate were monitored during HDL incubation. Over-expression of SR-BII, however, failed to promote the release of stored CE pools. In contrast, studies comparing HDL-stimulated efflux of free 3H-cholesterol from CHO-SR-BI/II cells showed that SR-BII is significantly better than SR-BI at cellular cholesterol efflux. I conclude that SR-BII has an important role in cellular cholesterol homeostasis and reverse cholesterol transport.

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