Abstract 392: The Role of Scavenger Receptor-BI’s Transmembrane Domains in Cholesterol Transport: A "Locked Dimer" Strategy

Abstract

Efficient reverse cholesterol transport requires interactions between high density lipoprotein (HDL) and its receptor, scavenger receptor-BI (SR-BI). SR-BI is an 82 kDa protein with a large extracellular domain anchored by two transmembrane domains (TMDs). Our lab recently solved the NMR structure of SR-BI’s C-terminal TMD (C-TMD), a region that mediates SR-BI dimerization. Further, FRET studies suggest HDL-induced movement between neighboring SR-BI monomers, which led to our hypothesis that flexibility between SR-BI TMDs facilitates cholesterol transport. Using structure-guided mutagenesis, we introduced cysteine residues into the C-TMD of full-length SR-BI to create “locked dimers” of the receptor. Total lysate and cell surface expression of WT-, A444C-, L451C-, or G453C-SR-BI were verified in transiently-transfected COS-7 cells by immunoblot analysis and flow cytometry, respectively. Based on the predicted orientation of sulfhydryl side chains relative to the putative dimerization motif, we used immunoblot analysis following electrophoresis under reducing/non-reducing conditions to confirm that A444C- and L451C-SR-BI, but not G453C-SR-BI, formed disulfide bonds. Compared to WT-SR-BI, the locked dimer mutants, A444C- and L451C-SR-BI, exhibited normal selective uptake of [ 3 H]-cholesteryl oleyl ether, despite slightly reduced [ 125 I]-HDL binding. SR-BI-mediated cholesterol efflux to HDL from cells pre-labeled with [ 3 H]-cholesterol was also unaltered by the presence of locked dimers. Finally, we investigated the ability of WT and mutant SR-BI receptors to alter accessibility of membrane free cholesterol to exogenous cholesterol oxidase (as judged by cholestenone levels). L451C- or G453C-SR-BI expression led to reduced cholestenone production compared to WT-SR-BI, suggesting that these mutants may be defective in reorganizing pools of membrane cholesterol. In conclusion, our preliminary data suggest that limiting conformational flexibility between TMDs by forcing locked dimers of SR-BI may not have a major impact on SR-BI-mediated cholesterol transport. However, locked dimers of SR-BI appear to affect the ability of SR-BI to modulate plasma membrane pools of free cholesterol, and this deserves further investigation.