Highly purified scavenger receptor class B,type I reconstituted into phosphatidylcholine/cholesterol liposomes mediates high affinity high density lipoprotein binding and selective lipid uptake

The murine class B, type I scavenger receptor mSR-BI is a high and low density lipoprotein (HDL and LDL) receptor that mediates selective uptake of cholesteryl esters. Here we describe a reconstituted phospholipid/cholesterol liposome assay of the binding and selective uptake activities of SR-BI derived from detergent-solubilized cells. The assay, employing lysates from epitope-tagged receptor (mSR-BI-t1)-expressing mammalian and insect cells, recapitulated many features of SR-BI activity in intact cells, including high affinity and saturable (125)I-HDL binding, selective lipid uptake from [(3)H]cholesteryl ether-labeled HDL, and poor inhibition of HDL receptor activity by LDL. The novel properties of a mutated receptor (Q402R/Q418R, normal LDL binding but loss of most HDL binding) were reproduced in the assay, as was the ability of the SR-BI homologue CD36 to bind HDL but not mediate efficient lipid uptake. In this assay, essentially homogeneously pure mSR-BI-t1, prepared by single-step immunoaffinity chromatography, mediated high affinity HDL binding and efficient selective lipid uptake from HDL. Thus, SR-BI-mediated HDL binding and selective lipid uptake are intrinsic properties of the receptor that do not require the intervention of other proteins or specific cellular structures or compartments.     

Regulation of SR-BI-mediated selective lipid uptake in Chinese hamster ovary-derived cells by protein kinase signaling pathways

Scavenger receptor, class B, type I (SR-BI) mediates binding and internalization of a variety of lipoprotein and nonlipoprotein ligands, including HDL. Studies in genetically engineered mice revealed that SR-BI plays an important role in HDL reverse cholesterol transport and protection against atherosclerosis. Understanding how SR-BI's function is regulated may reveal new approaches to therapeutic intervention in atherosclerosis and heart disease. We utilized a model cell system to explore pathways involved in SR-BI-mediated lipid uptake from and signaling in response to distinct lipoprotein ligands: the physiological ligand, HDL, and a model ligand, acetyl LDL (AcLDL). In Chinese hamster ovary-derived cells, murine SR-BI (mSR-BI) mediates lipid uptake via distinct pathways that are dependent on the lipoprotein ligand. Furthermore, HDL and AcLDL activate distinct signaling pathways. Finally, mSR-BI-mediated selective lipid uptake versus endocytic uptake are differentially regulated by protein kinase signaling pathways. The protein kinase C (PKC) activator PMA and the phosphatidyl inositol 3-kinase inhibitor wortmannin increase the degree of mSR-BI-mediated selective lipid uptake, whereas a PKC inhibitor has the opposite effect. These data demonstrate that SR-BI's selective lipid uptake activity can be acutely regulated by intracellular signaling cascades, some of which can originate from HDL binding to murine SR-BI itself.     

Analysis of chimeric receptors shows that multiple distinct functional activities of scavenger receptor, class B, type I (SR-BI), are localized to the extracellular receptor domain

Scavenger receptor BI (SR-BI) mediates the selective uptake of high-density lipoprotein (HDL) cholesteryl ester (CE), a process by which HDL CE is taken into the cell without degradation of the HDL particle. In addition, SR-BI stimulates the bi-directional flux of free cholesterol (FC) between cells and lipoproteins, an activity that may be responsible for net cholesterol efflux from peripheral cells as well as the rapid hepatic clearance of FC from plasma HDL. SR-BI also increases cellular cholesterol mass and alters cholesterol distribution in plasma membrane domains as judged by the enhanced sensitivity of membrane cholesterol to extracellular cholesterol oxidase. In contrast, CD36, a closely related class B scavenger receptor, has none of these activities despite binding HDL with high affinity. In the present study, analyses of chimeric SR-BI/CD36 receptors and domain-deleted SR-BI have been used to test the various domains of SR-BI for functional activities related to HDL CE selective uptake, bi-directional FC flux, and the alteration of membrane cholesterol mass and distribution. The results show that each of these activities localizes to the extracellular domain of SR-BI. The N-terminal cytoplasmic tail and transmembrane domains appear to play no role in these activities other than targeting the receptor to the plasma membrane. The C-terminal tail of SR-BI is dispensable for activity as well for targeting to the plasma membrane. Thus, multiple distinct functional activities are localized to the SR-BI extracellular domain.     

Extracellular hydrophobic regions in scavenger receptor BI play a key role in mediating HDL-cholesterol transport

The binding of high density lipoprotein (HDL) to scavenger receptor BI (SR-BI) is responsible for whole-body cholesterol disposal via reverse cholesterol transport. The extracellular domain of SR-BI is required for HDL binding and selective uptake of HDL-cholesterol. We identified six highly hydrophobic regions in this domain that may be important for receptor activity and performed site-directed mutagenesis to investigate the importance of these regions in SR-BI-mediated cholesterol transport. Non-conservative mutation of the regions encompassing V67, L140/L142, V164 or V221 reduced hydrophobicity and impaired the ability of SR-BI to bind HDL, mediate selective uptake of HDL-cholesterol, promote cholesterol efflux, and enlarge the cholesterol oxidase-sensitive pool of membrane free cholesterol. In contrast, conservative mutations at V67, V164 or V221 did not affect the hydrophobicity or these cholesterol transport activities. We conclude that the hydrophobicity of N-terminal extracellular regions of SR-BI is critical for cholesterol transport, possibly by mediating receptor-ligand and/or receptor-membrane interactions.     

Scavenger receptor class B, type I-mediated [3H]cholesterol efflux to high and low density lipoproteins is dependent on lipoprotein binding to the receptor

The murine scavenger receptor class B, type I (mSR-BI) is a receptor for high density lipoprotein (HDL), low density lipoprotein (LDL), and acetylated LDL (AcLDL). It mediates selective uptake of lipoprotein lipid and stimulates efflux of [(3)H]cholesterol to lipoproteins. SR-BI-mediated [(3)H]cholesterol efflux was proposed to be independent of ligand binding. In this study, using anti-mSR-BI antibody KKB-1 and two mSR-BI mutants with altered ligand binding properties, we demonstrated that SR-BI-mediated [(3)H]cholesterol efflux to lipoproteins was correlated with ligand binding and lipid uptake activities of the receptor. The KKB-1 antibody, which blocked lipoprotein binding without substantially altering the cholesterol oxidase-accessible cellular [(3)H]cholesterol, also blocked [(3)H]cholesterol efflux to HDL and LDL. One of the SR-BI mutants, which has a double substitution of arginines for glutamines at positions 402 and 418 (Q402R/Q418R), exhibited a high level of LDL binding and lipid uptake from LDL, but lost most of the corresponding HDL receptor activity. This mutant could mediate efficient [(3)H]cholesterol efflux to LDL, but not to HDL. Another mutant, M158R, with an arginine in place of methionine at position 158, exhibited reduced HDL and LDL receptor activities, but apparently normal AcLDL receptor activity. This mutant could mediate efficient [(3)H]cholesterol efflux to AcLDL, but not to HDL or LDL. These results suggest that SR-BI-stimulated [(3)H]cholesterol efflux to lipoproteins critically depends on ligand binding to this receptor and raise the possibility that the mechanisms of selective lipid uptake and [(3)H]cholesterol efflux may be intimately related.     

Scavenger receptor class B, type I-mediated uptake of various lipids into cells. Influence of the nature of the donor particle interaction with the receptor

Scavenger receptor (SR)-BI is the first molecularly defined receptor for high density lipoprotein (HDL) and it can mediate the selective uptake of cholesteryl ester into cells. To elucidate the molecular mechanisms by which SR-BI facilitates lipid uptake, we examined the connection between lipid donor particle binding and lipid uptake using kidney COS-7 cells transiently transfected with SR-BI. We systematically compared the uptake of [(3)H]cholesteryl oleoyl ether (CE) and [(14)C]sphingomyelin (SM) from apolipoprotein (apo) A-I-containing reconstituted HDL (rHDL) particles and apo-free lipid donor particles. Although both types of lipid donor could bind to SR-BI, only apo-containing lipid donors exhibited preferential delivery of CE over SM (i.e. nonstoichiometric lipid uptake). In contrast, apo-free lipid donor particles (phospholipid unilamellar vesicles, lipid emulsion particles) gave rise to stoichiometric lipid uptake due to interaction with SR-BI. This apparent whole particle uptake was not due to endocytosis, but rather fusion of the lipid components of the lipid donor with the cell plasma membrane; this process is perhaps mediated by a fusogenic motif in the extracellular domain of SR-BI. The interaction of apoA-I with SR-BI not only prevents fusion of the lipid donor with the plasma membrane but also allows the optimal selective lipid uptake. A comparison of rHDL particles containing apoA-I and apoE-3 showed that while both particles bound equally well to SR-BI, the apoA-I particle gave approximately 2-fold greater CE selective uptake. Catabolism of all major HDL lipids can occur via SR-BI with the relative selective uptake rate constants for CE, free cholesterol, triglycerides (triolein), and phosphatidylcholine being 1, 1.6, 0.7, and 0.2, respectively. It follows that a putative nonpolar channel created by SR-BI between the bound HDL particle and the cell plasma membrane is better able to accommodate the uptake of neutral lipids (e.g. cholesterol) relative to polar phospholipids.     

Elevated triglyceride content diminishes the capacity of high density lipoprotein to deliver cholesteryl esters via the scavenger receptor class B type I (SR-BI)

The selective uptake of high density lipoprotein (HDL) cholesteryl ester (CE) by the scavenger receptor class B type I (SR-BI) is well documented. However, the effect of altered HDL composition, such as occurs in hyperlipidemia, on this important process is not known. This study investigated the impact of variable CE and triglyceride (TG) content on selective uptake. CE selective uptake by Y1 and HepG2 cells was strongly affected by modification of either the CE or TG content of HDL. Importantly, TG, like CE, was selectively taken up by a dose-dependent, saturable process in these cells. As shown by ACTH up-regulation and receptor overexpression experiments, SR-BI mediated the selective uptake of both CE and TG. With in vitro modified HDLs of varying CE and TG composition, the selective uptake of CE and TG was dependent on the abundance of each lipid within the HDL particle. Furthermore, total selective uptake (CE + TG) remained constant, indicating that these lipids competed for cellular uptake. These data support a novel mechanism whereby SR-BI binds HDL and mediates the incorporation of a nonspecific portion of the HDL lipid core. In this way, TG directly affects the ability of HDL to donate CE to cells. Processes that raise the TG/CE ratio of HDL will impair the delivery of CE to cells via this receptor and may compromise the efficiency of sterol balancing pathways such as reverse cholesterol transport.     

Scavenger receptor class B type I-mediated cholesteryl ester-selective uptake and efflux of unesterified cholesterol. Influence of high density lipoprotein size and structure

Scavenger receptor (SR)-BI catalyzes the selective uptake of cholesteryl ester (CE) from high density lipoprotein (HDL) by a two-step process that involves the following: 1) binding of HDL to the receptor and 2) diffusion of the CE molecules into the cell plasma membrane. We examined the effects of the size of discoidal HDL particles containing wild-type (WT) apoA-I on selective uptake of CE and efflux of cellular free (unesterified) cholesterol (FC) from COS-7 cells expressing SR-BI to determine the following: 1) the influence of apoA-I conformation on the lipid transfer process, and 2) the contribution of receptor binding-dependent processes to the overall efflux of cellular FC. Large (10 nm diameter) reconstituted HDL bound to SR-BI better (B(max) approximately 420 versus 220 ng of apoA-I/mg cell protein), delivered more CE, and promoted more FC efflux than small ( approximately 8 nm) particles. When normalized to the number of reconstituted HDL particles bound to the receptor, the efficiencies of either CE uptake or FC efflux with these particles were the same indicating that altering the conformation of WT apoA-I modulates binding to the receptor (step 1) but does not change the efficiency of the subsequent lipid transfer (step 2); this implies that binding induces an optimal alignment of the WT apoA-I.SR-BI complex so that the efficiency of lipid transfer is always the same. FC efflux to HDL is affected both by binding of HDL to SR-BI and by the ability of the receptor to perturb the packing of FC molecules in the cell plasma membrane.     

Cellular and physiological roles of SR-BI, a lipoprotein receptor which mediates selective lipid uptake

High-density lipoproteins (HDL) play an important role in protection against atherosclerosis by mediating reverse cholesterol transport - the transport of excess cholesterol from peripheral tissues to the liver for disposal. SR-BI is a cell surface receptor for HDL and other lipoproteins (LDL and VLDL) and mediates the selective uptake of lipoprotein cholesterol by cells. Overexpression or genetic ablation of SR-BI in mice revealed that it plays an important role in HDL metabolism and reverse cholesterol transport and protects against atherosclerosis in mouse models of the disease. If it plays a similar role in humans then it may be an attractive target for therapeutic intervention. We will review some of the recent advances in the understanding of SR-BI's physiological role and cellular function in lipoprotein metabolism.     

SR-BI的分子结构及其表达调控

小鼠B族Ⅰ型清道夫受体是目前已确认的唯一真正介导细胞与高密度脂蛋白作用的膜受体,主要在肝脏和固醇生成组织中表达,并受促激素、胆固醇、饮食以及药理等因素所调控。该受体介导高密度脂蛋白-胆固醇酯的选择性吸收,是调节胆固醇逆转运的唯一靶点,在高密度脂蛋白代谢和胆固醇运输中起重要作用。该基因缺陷对不同的组织具有不同的影响。它有可能作为一个新的治疗靶点来预防和治疗动脉粥样硬化性心脑血管疾病。对其分子结构、表达调控及相关研究作了详细介绍。     

Extracellular disulfide bonds support scavenger receptor class B type I-mediated cholesterol transport

Scavenger receptor class B type I (SR-BI) binds high-density lipoprotein (HDL) and mediates the selective uptake of cholesteryl esters (CE). Although the extracellular domain of SR-BI is critical for function, the structural characteristics of this region remain elusive. Using sulfhydryl labeling strategies, we report the novel finding that all six cysteine (Cys) residues in the extracellular domain of SR-BI are involved in disulfide bond formation that is intramolecular by nature. We hypothesized that an SR-BI conformation stabilized by extracellular disulfide bonds is a prerequisite for SR-BI-mediated cholesterol transport. Thus, single-Cys mutant SR-BI receptors (C251S-, C280S-, C321S-, C323S-, C334S-, and C384S-SR-BI), as well as Cys-less SR-BI, a mutant SR-BI receptor void of all Cys residues, were created, and plasma membrane localization was confirmed. Functional assays revealed that C280S-, C321S-, C323S-, and C334S-SR-BI and Cys-less SR-BI mutant receptors displayed weakened HDL binding and subsequent selective uptake of HDL-CE. However, only C323S-SR-BI and Cys-less SR-BI were unable to mediate wild-type levels of efflux of free cholesterol (FC) to HDL. None of the Cys mutations disrupted SR-BI's ability to redistribute plasma membrane FC. Taken together, the intramolecular disulfide bonds in the extracellular domain of SR-BI appear to maintain the receptor in a conformation integral to its cholesterol transport functions.     

Overexpression of SR-BI by adenoviral vector promotes clearance of apoA-I,but not apoB,in human apoB transgenic mice

Scavenger receptor BI (SR-BI) is a multi-ligand lipoprotein receptor that mediates selective lipid uptake from HDL, and plays a central role in hepatic HDL metabolism. In this report, we investigated the extent to which SR-BI selective lipid uptake contributes to LDL metabolism. As has been reported for human LDL, mouse SR-BI expressed in transfected cells mediated selective lipid uptake from mouse LDL. However, LDL-cholesteryl oleoyl ester (CE) transfer relative to LDL-CE bound to the cell surface (fractional transfer) was approximately 18-fold lower compared with HDL-CE. Adenoviral vector-mediated SR-BI overexpression in livers of human apoB transgenic mice ( approximately 10-fold increased expression) reduced plasma HDL-cholesterol (HDL-C) and apolipoprotein (apo)A-I concentrations to nearly undetectable levels 3 days after adenovirus infusion. Increased hepatic SR-BI expression resulted in only a modest depletion in LDL-C that was restricted to large LDL particles, and no change in steady-state concentrations of human apoB. Kinetic studies showed a 19% increase in the clearance rate of LDL-CE in mice with increased SR-BI expression, but no change in LDL apolipoprotein clearance. Quantification of hepatic uptake of LDL-CE and LDL-apolipoprotein showed selective uptake of LDL-CE in livers of human apo B transgenic mice. However, such uptake was not significantly increased in mice over-expressing SR-BI. We conclude that SR-BI-mediated selective uptake from LDL plays a minor role in LDL metabolism in vivo.     

The fate of HDL particles in vivo after SR-BI-mediated selective lipid uptake

Scavenger receptor class B type I (SR-BI) delivers cholesterol ester from HDL to cells via a selective uptake mechanism, whereby lipid is transferred from the core of the particle without concomitant degradation of the protein moiety. The precise metabolic fate of HDL particles after selective lipid uptake is not known. To characterize SR-BI-mediated HDL processing in vivo, we expressed high levels of this receptor in livers of apoA-I(-/-) mice by adenoviral vector gene transfer, and then injected the mice with a bolus of human HDL(2) traced with (125)I-dilactitol tyramine. HDL recovered from apoA-I(-/-) mice over-expressing SR-BI was significantly smaller than HDL recovered from control mice as measured by non-denaturing gel electrophoresis. When injected into C57BL/6 mice, these HDL "remnants" were rapidly converted to HDL(2)-sized lipoprotein particles, and were cleared from the plasma at a rate similar to HDL(2). In assays in cultured cells, HDL remnants did not stimulate ATP-binding cassette transporter A1-dependent cholesterol efflux. When mixed with mouse plasma ex vivo, HDL remnants rapidly converted to larger HDL particles. These studies identify a previously ill-defined pathway in HDL metabolism, whereby SR-BI generates small, dense HDL particles that are rapidly remodeled in plasma. This remodeling pathway may represent a process that is important in determining the rate of apoA-I catabolism and HDL-mediated reverse cholesterol transport.     

Apolipoprotein A-II modulates the binding and selective lipid uptake of reconstituted high density lipoprotein by scavenger receptor BI

High density lipoprotein (HDL) represents a mixture of particles containing either apoA-I and apoA-II (LpA-I/A-II) or apoA-I without apoA-II (LpA-I). Differences in the function and metabolism of LpA-I and LpA-I/A-II have been reported, and studies in transgenic mice have suggested that apoA-II is pro-atherogenic in contrast to anti-atherogenic apoA-I. The molecular basis for these observations is unclear. The scavenger receptor BI (SR-BI) is an HDL receptor that plays a key role in HDL metabolism. In this study we investigated the abilities of apoA-I and apoA-II to mediate SR-BI-specific binding and selective uptake of cholesterol ester using reconstituted HDLs (rHDLs) that were homogeneous in size and apolipoprotein content. Particles were labeled in the protein (with (125)I) and in the lipid (with [(3)H]cholesterol ether) components and SR-BI-specific events were analyzed in SR-BI-transfected Chinese hamster ovary cells. At 1 microg/ml apolipoprotein, SR-BI-mediated cell association of palmitoyloleoylphosphatidylcholine-containing AI-rHDL was significantly greater (3-fold) than that of AI/AII-rHDL, with a lower K(d) and a higher B(max) for AI-rHDL as compared with AI/AII-rHDL. Unexpectedly, selective cholesterol ester uptake from AI/AII-rHDL was not compromised compared with AI-rHDL, despite decreased binding. The efficiency of selective cholesterol ester uptake in terms of SR-BI-associated rHDL was 4-5-fold greater for AI/AII-rHDL than AI-rHDL. These results are consistent with a two-step mechanism in which SR-BI binds ligand and then mediates selective cholesterol ester uptake with an efficiency dependent on the composition of the ligand. ApoA-II decreases binding but increases selective uptake. These findings show that apoA-II can exert a significant influence on selective cholesterol ester uptake by SR-BI and may consequently influence the metabolism and function of HDL, as well as the pathway of reverse cholesterol transport.     

Remodeling of HDL by CETP in vivo and by CETP and hepatic lipase in vitro results in enhanced uptake of HDL CE by cells expressing scavenger receptor B-I.

The transport of HDL cholesteryl esters (CE) from plasma to the liver involves a direct uptake pathway, mediated by hepatic scavenger receptor B-I (SR-BI), and an indirect pathway, involving the exchange of HDL CE for triglycerides (TG) of TG-rich lipoproteins by cholesteryl ester transfer protein (CETP). We carried out HDL CE turnover studies in mice expressing human CETP and/or human lecithin:cholesterol acyltransferase (LCAT) transgenes on a background of human apoA-I expression. The fractional clearance of HDL CE by the liver was delayed by LCAT transgene, while the CETP transgene increased it. However, there was no incremental transfer of HDL CE radioactivity to the TG-rich lipoprotein fraction in mice expressing CETP, suggesting increased direct removal of HDL CE in the liver. To evaluate the possibility that this might be mediated by SR-BI, HDL isolated from plasma of the different groups of transgenic mice was incubated with SR-BI transfected or control CHO cells. HDL isolated from mice expressing CETP showed a 2- to 4-fold increase in SR-BI-mediated HDL CE uptake, compared to HDL from mice lacking CETP. The addition of pure CETP to HDL in cell culture did not lead to increased selective uptake of HDL CE by cells. However, when human HDL was enriched with TG by incubation with TG-rich lipoproteins in the presence of CETP, then treated with hepatic lipase, there was a significant enhancement of HDL CE uptake. Thus, the remodeling of human HDL by CETP, involving CE;-TG interchange, followed by the action of hepatic lipase (HL), leads to the enhanced uptake of HDL CE by cellular SR-BI.These observations suggest that in animals such as humans in which both the selective uptake and CETP pathways are active, the two pathways could operate in a synergistic fashion to enhance reverse cholesterol transport.     

Serum amyloid A is a ligand for scavenger receptor class B type I and inhibits high density lipoprotein binding and selective lipid uptake

Serum amyloid A is an acute phase protein that is carried in the plasma largely as an apolipoprotein of high density lipoprotein (HDL). In this study we investigated whether SAA is a ligand for the HDL receptor, scavenger receptor class B type I (SR-BI), and how SAA may influence SR-BI-mediated HDL binding and selective cholesteryl ester uptake. Studies using Chinese hamster ovary cells expressing SR-BI showed that (125)I-labeled SAA, both in lipid-free form and in reconstituted HDL particles, functions as a high affinity ligand for SR-BI. SAA also bound with high affinity to the hepatocyte cell line, HepG2. Alexa-labeled SAA was shown by fluorescence confocal microscopy to be internalized by cells in a SR-BI-dependent manner. To assess how SAA association with HDL influences HDL interaction with SR-BI, SAA-containing HDL was isolated from mice overexpressing SAA through adenoviral gene transfer. SAA presence on HDL had little effect on HDL binding to SR-BI but decreased (30-50%) selective cholesteryl ester uptake. Lipid-free SAA, unlike lipid-free apoA-I, was an effective inhibitor of both SR-BI-dependent binding and selective cholesteryl ester uptake of HDL. We have concluded that SR-BI plays a key role in SAA metabolism through its ability to interact with and internalize SAA and, further, that SAA influences HDL cholesterol metabolism through its inhibitory effects on SR-BI-mediated selective lipid uptake.     

The apolipoprotein E-dependent low density lipoprotein cholesteryl ester selective uptake pathway in murine adrenocortical cells involves chondroitin sulfate proteoglycans and an alpha 2-macroglobulin receptor

Cells acquire lipoprotein cholesterol by receptor-mediated endocytosis and selective uptake pathways. In the latter case, lipoprotein cholesteryl ester (CE) is transferred to the plasma membrane without endocytosis and degradation of the lipoprotein particle. Previous studies with Y1/E/tet/2/3 murine adrenocortical cells that were engineered to express apolipoprotein (apo) E demonstrated that apoE expression enhances low density lipoprotein (LDL) CE uptake by both selective and endocytic pathways. The present experiments test the hypothesis that apoE-dependent LDL CE selective uptake is mediated by scavenger receptor, class B, type I (SR-BI). Surprisingly, SR-BI expression was not detected in the Y1/E/tet/2/3 clone of Y1 adrenocortical cells, indicating the presence of a distinct apoE-dependent pathway for LDL CE selective uptake. ApoE-dependent LDL CE selective uptake in Y1/E/tet/2/3 cells was inhibited by receptor-associated protein and by activated alpha(2)-macroglobulin (alpha(2)M), suggesting the participation of the LDL receptor-related protein/alpha(2)M receptor. Reagents that inhibited proteoglycan synthesis or removed cell surface chondroitin sulfate proteoglycan completely blocked apoE-dependent LDL CE selective uptake. None of these reagents inhibited SR-BI-mediated LDL CE selective uptake in the Y1-BS1 clone of Y1 cells in which LDL CE selective uptake is mediated by SR-BI. We conclude that LDL CE selective uptake in adrenocortical cells occurs via SR-BI-independent and SR-BI-dependent pathways. The SR-BI-independent pathway is an apoE-dependent process that involves both chondroitin sulfate proteoglycans and an alpha(2)M receptor.     

Separation of lipid transport functions by mutations in the extracellular domain of scavenger receptor class B,type I

Scavenger receptor class B, type I (SR-BI) shows a variety of effects on cellular cholesterol metabolism, including increased selective uptake of high density lipoprotein (HDL) cholesteryl ester, stimulation of free cholesterol (FC) efflux from cells to HDL and phospholipid vesicles, and changes in the distribution of plasma membrane FC as evidenced by increased susceptibility to exogenous cholesterol oxidase. Previous studies showed that these multiple effects require the extracellular domain of SR-BI, but not the transmembrane and cytoplasmic domains. To test whether 1) the extracellular domain of SR-BI mediates multiple activities by virtue of discrete functional subdomains, or 2) the multiple activities are, in fact, secondary to and driven by changes in cholesterol flux, the extracellular domain of SR-BI was subjected to insertional mutagenesis by strategically placing an epitope tag into nine sites. These experiments identified four classes of mutants with disruptions at different levels of function. Class 4 mutants showed a clear separation of function between HDL binding, HDL cholesteryl ester uptake, and HDL-dependent FC efflux on one hand and FC efflux to small unilamellar vesicles and an increased cholesterol oxidase-sensitive pool of membrane FC on the other. Selective disruption of the latter two functions provides evidence for multiple functional subdomains in the extracellular receptor domain. Furthermore, these findings uncover a difference in the SR-BI-mediated efflux pathways for FC transfer to HDL acceptors versus phospholipid vesicles. The loss of the cholesterol oxidase-sensitive FC pool and FC efflux to small unilamellar vesicle acceptors in Class 4 mutants suggests that these activities may be mechanistically related.     

Glycine dimerization motif in the N-terminal transmembrane domain of the high density lipoprotein receptor SR-BI required for normal receptor oligomerization and lipid transport

Scavenger receptor class B, type I (SR-BI), a CD36 superfamily member, is an oligomeric high density lipoprotein (HDL) receptor that mediates negatively cooperative HDL binding and selective lipid uptake. We identified in the N-terminal transmembrane (N-TM) domain of SR-BI a conserved glycine dimerization motif, G(15)X(2)G(18)X(3)AX(2)G(25), of which the submotif G(18)X(3)AX(2)G(25) significantly contributes to homodimerization and lipid uptake activity. SR-BI variants were generated by mutations (single or multiple Gly → Leu substitutions) or by replacing the N-TM domain with those from other CD36 superfamily members containing (croquemort) or lacking (lysosomal integral membrane protein (LIMP) II) this glycine motif (chimeras). None of the SR-BI variants exhibited altered surface expression (based on antibody binding) or HDL binding. However, the G15L/G18L/G25L triple mutant exhibited reductions in cell surface homo-oligomerization (>10-fold) and the rate of selective lipid uptake (～ 2-fold). Gly(18) and Gly(25) were necessary for normal lipid uptake activity of SR-BI and the SR-BI/croquemort chimera. The lipid uptake activity of the glycine motif-deficient SR-BI/LIMP II chimera was low but could be increased by introducing glycines at positions 18 and 25. The rate of lipid uptake mediated by SR-BI/LIMP II chimeras was proportional to the extent of receptor oligomerization. Thus, the glycine dimerization motif G(18)X(3)AX(2)G(25) in the N-TM domain of SR-BI contributes substantially to the homo-oligomerization and lipid transport activity of SR-BI but does not influence the negative cooperativity of HDL binding. Oligomerization-independent binding cooperativity suggests that classic allostery is not involved and that the negative cooperativity is probably the consequence of a "lattice effect" (interligand steric interference accompanying binding to adjacent receptors).     

Dissociation of the high density lipoprotein and low density lipoprotein binding activities of murine scavenger receptor class B type I (mSR-BI) using retrovirus library-based activity dissection

The murine class B, type I scavenger receptor (mSR-BI) is a receptor for both high density lipoprotein (HDL) and low density lipoprotein (LDL) and mediates selective, rather than endocytic, uptake of lipoprotein lipid. We have developed a "retrovirus library-based activity dissection" method to generate mSR-BI mutants in which some, but not all, of the activities of this multifunctional protein have been disrupted. This method employs three techniques: 1) efficient in vitro cDNA mutagenesis (here error-prone PCR was used), 2) efficient retroviral delivery and high expression of single mutant cDNAs into individual cells, and 3) isolation of infected cells expressing the desired mutant phenotype using high sensitivity positive/negative screening by two-color fluorescence-activated cell sorting. A set of mutants, all having arginine substitutions at two common sites (positions 402 or 401 and position 418), were isolated and characterized. Mutation at either site alone did not generate as strong a mutant phenotype (loss of DiI uptake from DiI-HDL) as did the double mutations. "Activity-dissected" double mutants were as effective as wild-type mSR-BI in functioning as LDL receptors, mediating high affinity LDL binding and uptake of metabolically active cholesterol from LDL, but they lost most of their corresponding HDL receptor activity. Thus, these mutants provide support for the proposal that the interaction of SR-BI with HDL differs from that with LDL. Examination of the in vivo function of such mutants may provide insights into the differential roles of the LDL and HDL receptor activities of SR-BI in normal lipoprotein metabolism and in SR-BI's ability to protect against atherosclerosis.