Abstract 522: Scavenger Receptor-BI’s Cholesterol Transport Functions are Dependent on its Extracellular Tryptophan Residues

Abstract

High density lipoprotein (HDL) functions as an anti-atherogenic particle, primarily due to its role in reverse cholesterol transport whereby HDL delivers cholesterol to the liver for excretion upon interaction with its receptor, scavenger receptor BI (SR-BI). The extracellular domain of SR-BI is required for its cholesterol transport functions, yet our understanding of the molecular and structural features of this domain remains limited. We designed experiments to test the hypothesis that one or more of the six highly conserved extracellular tryptophan (Trp; W) residues are critical for mediating receptor function. Towards this end, we created a series of Trp-to-Phe mutant receptors of SR-BI, as well as Trp-free SR-BI and assessed the ability of these mutant receptors to mediate cholesterol transport. Wild-type (WT) or mutant SR-BI receptors were transiently expressed in COS7 cells and proper cell surface expression was confirmed by immunoblotting, confocal microscopy and flow cytometry. Next, we showed that Trp-free- and W415F-SR-BI had a significantly decreased ability to bind HDL (12.7% and 31.3% of WT levels, respectively) and promote selective uptake of HDL-cholesteryl esters (35.2% and 70.1% of WT levels, respectively). Interestingly, only Trp-free-, but not W415F-SR-BI, showed an impaired ability to mediate efflux of free cholesterol (FC) (90.8% decrease vs. WT). Furthermore, both W415F- and Trp-free SR-BI were unable to reorganize plasma membrane pools of FC based on lack of sensitivity of FC to exogenous cholesterol oxidase. We then designed an additional set of mutant SR-BI receptors to determine whether restoration of Trp415 alone (or in combination with other Trp residues) could rescue SR-BI function. Restoration of Trp415 into Trp-free-SR-BI partially rescued cholesterol transport functions. Addition of any of the other 5 extracellular Trp residues was also not sufficient to restore WT cholesterol transport function in combination with Trp415. In summary, loss of all Trp residues in SR-BI impairs its cholesterol transport functions, mostly due to the loss of Trp415. Homology modeling of SR-BI based on the crystal structure of LIMP-2, a member of the same protein family, may help identify the importance of this residue in future studies.