High density lipoprotein (HDL) and low density lipoprotein (LDL) are cholesterol transport particles whose plasma concentrations are directly (LDL) and inversely (HDL) correlated with risk for atherosclerosis. LDL catabolism involves cellular uptake and degradation of the entire particle by a well-characterized receptor. HDL, in contrast, selectively delivers its cholesterol, but not protein, to cells by unknown receptors. Here it is shown that the class B scavenger receptor SR-BI is an HDL receptor. SR-BI binds HDL with high affinity, is expressed primarily in liver and nonplacental steroidogenic tissues, and mediates selective cholesterol uptake by a mechanism distinct from the classic LDL receptor pathway.

The class B, type I scavenger receptor, SR-BI, was the first molecularly well defined cell surface high density lipoprotein (HDL) receptor to be described. It mediates transfer of lipid from HDL to cells via selective lipid uptake, a mechanism distinct from receptor-mediated endocytosis via clathrin-coated pits and vesicles. SR-BI is expressed most abundantly in steroidogenic tissues (adrenal gland, ovary), where trophic hormones coordinately regulate its expression with steroidogenesis, and in the liver, where it may participate in reverse cholesterol transport. Here we have used immunochemical methods to study the structure and subcellular localization of murine SR-BI (mSR-BI) expressed either in transfected Chinese hamster ovary cells or in murine adrenocortical Y1-BS1 cells. mSR-BI, an ϳ82-kDa glycoprotein, was initially synthesized with multiple high mannose N-linked oligosaccharide chains, and some, but not all, of these were processed to complex forms during maturation of the protein in the Golgi apparatus. Metabolic labeling with [ 3 H]palmitate and [ 3 H]myristate demonstrated that mSR-BI was fatty acylated, a property shared with CD36, another class B scavenger receptor, and other proteins that concentrate in specialized, cholesterol-and glycolipid-rich plasma membrane microdomains called caveolae. OptiPrep density gradient fractionation of plasma membranes established that mSR-BI copurified with caveolin-1, a constituent of caveolae; and immunofluorescence microscopy demonstrated that mSR-BI colocalized with caveolin-1 in punctate microdomains across the surface of cells and on the edges of cells. Thus, mSR-BI colocalizes with caveolae, and this raises the possibility that the unique properties of these specialized cell surface domains may play a critical role in SR-BI-mediated transfer of lipids between lipoproteins and cells. High density lipoprotein (HDL)1 and low density lipoprotein (LDL) play important roles in the movement of cholesterol and cholesteryl esters through the plasma and in receptor-mediated sterol uptake by tissues. The mechanisms by which HDL delivers cholesterol and cholesteryl ester to steroidogenic (3) and hepatic (4) cells, although not well understood, clearly differ from the classic LDL receptor pathway, which involves clathrin-coated pits, coated vesicles, endosomes, and lysosomes (1, 2). HDL particles bind to cell surface receptors, but the intact particles are neither transported to lysosomes nor degraded (hydrolyzed). Instead, the core cholesteryl esters are selectively transferred into cells, and the lipid-depleted apolipoprotein-containing shell is released into the extracellular fluid, a process known as selective cholesterol uptake (5-11). The detailed molecular mechanisms underlying selective cholesterol uptake from HDL have not yet been determined.Recent in vitro and in vivo studies have suggested that the class B, type I scavenger receptor, SR-BI, is a cell surface HDL receptor that mediates physiologically relevant selective cholesterol uptake (12)(13)(14). SR-BI ...

The class B type I scavenger receptor, (SR-BI), is a member of the CD36 superfamily of proteins and is a physiologically relevant, high affinity cell surface high density lipoprotein (HDL) receptor that mediates selective lipid uptake. The mechanism of selective lipid uptake is fundamentally different from that of classic receptor-mediated uptake via coated pits and vesicles (e.g. the low density lipoprotein receptor pathway) in that it involves efficient transfer of the lipids, but not the outer shell proteins, from HDL to cells. The abilities of SR-BI and CD36, both of which are class B scavenger receptors, to bind HDL and mediate cellular uptake of HDLassociated lipid when transiently expressed in COS cells were examined. For these experiments, the binding of HDL to cells was assessed using either 125 I-or Alexa (a fluorescent dye)-HDL in which the apolipoproteins on the surface of the HDL particles were covalently modified. Lipid transfer was measured using HDL noncovalently labeled by the fluorescent lipid 1,1-dioctadecyl-3,3,3,3-tetramethylindocarbocyanine perchlorate. Although both mSR-BI and human CD36 (hCD36) could mediate the binding of HDL in a punctate pattern across the surfaces of cells, only mSR-BI efficiently mediated the transfer of lipid to the cells. Analysis of point mutants established that the major sites of fatty acylation of mSR-BI are Cys 462 and Cys 470 and that fatty acylation is not required for receptor clustering, HDL binding, or efficient lipid transfer. Generation of mSR-BI/hCD36 domain swap chimeras showed that the differences in lipid uptake activities between mSR-BI and hCD36 were not due to differences between their two sets of transmembrane and cytoplasmic domains but rather result from differences in their large extracellular loop domains. These results show that high affinity binding to a cell surface receptor is not sufficient to ensure efficient cellular lipid uptake from HDL. Thus, SR-BI-mediated binding combined with SR-BI-dependent facilitated transfer of lipid from the HDL particle to the cell appears to be the most likely mechanism for the bulk of the selective uptake of cholesteryl esters from HDL to the liver and steroidogenic tissues.The protective role of HDL 1 against cardiovascular disease is commonly attributed to its ability to remove excess cholesterol from cells in the arterial wall and transport it to the liver for disposal, a process called reverse cholesterol transport (1, 2). In addition to delivering cholesterol to the liver, HDL has been shown to be a significant source of cholesterol both for steroidogenesis and for cholesterol storage depots in steroidogenic tissues (adrenal gland, ovary, testis) and cells (3-11). The novel mechanism by which the cholesteryl esters in HDL are transferred to liver and steroidogenic tissues is called selective uptake (Refs. 12-18; reviewed in Refs. 9 and 19) and is distinct from the classic receptor-mediated pathway of lipoprotein particle endocytosis via coated pits and vesicles to lysosomes (20). The first step of s...

The binding of apoA-I-containing ligands to the HDL receptor scavenger receptor class B type I (SR-BI) was characterized using two different assays. The first employed conventional binding or competition assays with 125 I-labeled ligands. The second is a new nonradioactive ligand binding assay, in which the receptor-associated ligand is detected by quantitative immunoblotting ("immunoreceptor assay"). Using both methods, we observed that the K d value for spherical HDL (density ‫؍‬ 1.1-1.13 g/ml) was ϳ16 g of protein/ml, while the values for discoidal reconstituted HDL (rHDL) containing proapoA-I or plasma apoA-I were substantially lower (ϳ0.4 -5 g of protein/ml). We also observed reduced affinity and/or competition for spherical 125 I-HDL cell association by higher relative to lower density HDL and very poor competition by lipid-free apoA-I and pre-␤-1 HDL. Deletion of either 58 carboxyl-terminal or 59 amino-terminal residues from apoA-I, relative to full-length control apoA-I, resulted in little or no change in the affinity of corresponding rHDL particles. However, rHDL particles containing a double mutant lacking both terminal domains competed poorly with spherical 125 I-HDL for binding to SR-BI. These findings suggest an important role for apoA-I and its conformation/organization within particles in mediating HDL binding to SR-BI and indicate that the NH 2 and COOH termini of apoA-I directly or indirectly contribute independently to binding to SR-BI.

The HDL receptor, scavenger receptor, class B, type I (SR-BI), is a homooligomeric cell surface glycoprotein that controls HDL structure and metabolism by mediating the cellular selective uptake of lipids, mainly cholesteryl esters, from HDL. The mechanism underlying SR-BI-mediated lipid transfer, which differs from classic receptor-mediated endocytosis, involves a two-step process (binding followed by lipid transport) that is poorly understood. Our previous structure/activity analysis of the small-molecule inhibitor blocker of lipid transport 1 (BLT-1), which potently (IC 50 ∼ 50 nM) blocks SR-BI-mediated lipid transport, established that the sulfur in BLT-1's thiosemicarbazone moiety was essential for activity. Here we show that BLT-1 is an irreversible inhibitor of SR-BI, raising the possibility that cysteine(s) in SR-BI interact with BLT-1. Mass spectrometric analysis of purified SR-BI showed two of its six exoplasmic cysteines have free thiol groups (Cys251 and Cys384). Converting Cys384 (but not Cys251) to serine resulted in complete BLT-1 insensitivity, establishing that the unique molecular target of BLT-1 inhibition of cellular SR-BI dependent lipid transport is SR-BI itself. The C384S substitution reduced the receptor's intrinsic lipid uptake activity by approximately 60% without dramatically altering its surface expression, homooligomerization, or HDL binding. Thus, a small-molecule screening approach identified a key residue in SR-BI involved in lipid transport, providing a powerful springboard into the analyses of the structure and mechanism of SR-BI, and highlighting the power of this approach for such analyses.