Abstract 72: Tryptophan 415 of Scavenger Receptor BI Is Critical for Mediating Cholesterol Transport Functions

Abstract

High density lipoproteins (HDL) combat atherosclerosis by delivering excess cholesteryl esters (CE) from peripheral tissues to the liver for excretion upon binding to their receptor, scavenger receptor BI (SR-BI), via reverse cholesterol transport. Our previous studies demonstrate that the extracellular domain (ECD) of SR-BI, particularly areas of hydrophobicity in its N-terminal half, is critical for cholesterol transport functions. The ECD of SR-BI also contains eight tryptophan (Trp;W) residues that may play a role in positioning SR-BI within the membrane, as observed with other membrane proteins. Based on their location at the membrane-water interface, we hypothesize that three of these Trp residue (W9, W56, W415), are critical for SR-BI’s cholesterol transport functions. To test this hypothesis, we designed single Trp-to-Phe mutations, as well as a Trp-free-SR-BI and confirmed their expression upon transient transfection into COS7 cells. Next, we used [ 3 H]/[ 125 I]-HDL-COE to assess the ability of these receptors to bind HDL. Our data revealed that Trp-free-SR-BI and W415F-SR-BI had a significantly decreased ability (88% and 63%, respectively) to bind HDL compared to wild-type (WT) SR-BI, accompanied by 44 and 64% decreases, respectively, in the ability to mediate selective uptake of HDL-COE. Trp-free- and W415F-SR-BI were also unable to enlarge the cholesterol oxidase-sensitive pool of membrane free cholesterol (FC) (20-50% decreases), as compared to WT SR-BI. The inability of these two mutant receptors to mediate these functions was not due to impaired trafficking, as both receptors were present on the cell surface as judged by flow cytometry and immunofluorescence studies. Interestingly, only Trp-free-SR-BI was unable to mediate the release of FC out of cells (50% decrease compared to WT SR-BI). Together, these data suggest that Trp415, that flanks a transmembrane segment of SR-BI, might be involved in orienting SR-BI in a conformation that would support HDL binding and selective uptake of HDL-CE, as well as reorganization of plasma membrane cholesterol. We are currently identifying the combination of Trp residues at the membrane/water interface that are important for mediating FC efflux to HDL.