Plasma high density lipoprotein (HDL), which protects against atherosclerosis, is thought to remove cholesterol from peripheral tissues and to deliver cholesteryl esters via a selective uptake pathway to the liver (reverse cholesterol transport) and steroidogenic tissues (e.g., adrenal gland for storage and hormone synthesis). Despite its physiologic and pathophysiologic importance, the cellular metabolism of HDL has not been well defined. The class B, type I scavenger receptor (SR-BI) has been proposed to play an important role in HDL metabolism because (i) it is a cell surface HDL receptor which mediates selective cholesterol uptake in cultured cells, (ii) its physiologically regulated expression is most abundant in the liver and steroidogenic tissues, and (iii) hepatic overexpression dramatically lowers plasma HDL. To test directly the normal role of SR-BI in HDL metabolism, we generated mice with a targeted null mutation in the SR-BI gene. In heterozygous and homozygous mutants relative to wild-type controls, plasma cholesterol concentrations were increased by Ϸ31% and 125%, respectively, because of the formation of large, apolipoprotein A-I (apoA-I)-containing particles, and adrenal gland cholesterol content decreased by 42% and 72%, respectively. The plasma concentration of apoA-I, the major protein in HDL, was unchanged in the mutants. This, in conjunction with the increased lipoprotein size, suggests that the increased plasma cholesterol in the mutants was due to decreased selective cholesterol uptake. These results provide strong support for the proposal that in mice the gene encoding SR-BI plays a key role in determining the levels of plasma lipoprotein cholesterol (primarily HDL) and the accumulation of cholesterol stores in the adrenal gland. If it has a similar role in controlling plasma HDL in humans, SR-BI may inf luence the development and progression of atherosclerosis and may be an attractive candidate for therapeutic intervention in this disease.

The high density lipoprotein (HDL) receptor SR-BI (scavenger receptor class B type I) mediates the selective uptake of plasma HDL cholesterol by the liver and steroidogenic tissues. As a consequence, SR-BI can inf luence plasma HDL cholesterol levels, HDL structure, biliary cholesterol concentrations, and the uptake, storage, and utilization of cholesterol by steroid hormone-producing cells. Here we used homozygous null SR-BI knockout mice to show that SR-BI is required for maintaining normal biliary cholesterol levels, oocyte development, and female fertility. We also used SR-BI͞apolipoprotein E double homozygous knockout mice to show that SR-BI can protect against early-onset atherosclerosis. Although the mechanisms underlying the effects of SR-BI loss on reproduction and atherosclerosis have not been established, potential causes include changes in (i) plasma lipoprotein levels and͞or structure, (ii) cholesterol f lux into or out of peripheral tissues (ovary, aortic wall), and (iii) reverse cholesterol transport, as indicated by the significant reduction of gallbladder bile cholesterol levels in SR-BI and SR-BI͞apolipoprotein E double knockout mice relative to controls. If SR-BI has similar activities in humans, it may become an attractive target for therapeutic intervention in a variety of diseases.High density lipoprotein (HDL)-cholesterol levels are inversely proportional to the risk for atherosclerosis (1). This may be due partly to ''reverse cholesterol transport'' (RCT), in which HDL is proposed to remove excess cholesterol from cells, including those in the artery wall (2-7), and transport it, either indirectly or directly (8, 9), to the liver for biliary secretion. HDL also can deliver cholesterol directly to steroidogenic tissues (adrenal gland, testis, ovary) for storage in cytoplasmic cholesteryl ester droplets and for steroid hormone synthesis (10-12). Thus, HDL may influence a variety of endocrine functions, including reproduction. A key mechanism of receptor-mediated direct delivery of HDL cholesteryl esters to the liver and steroidogenic tissues is selective cholesterol uptake, in which only the cholesteryl esters of the HDL particles (not the apolipoproteins) are transferred efficiently to cells (8, 9).The class B type I scavenger receptor, SR-BI, is a cellsurface HDL receptor that mediates selective lipid uptake (13-21; reviewed in refs. 22 and 23). It is most highly expressed in the liver and steroidogenic tissues, in which its activity is regulated by trophic hormones (13, 24-31). As a consequence, SR-BI is a key regulator of HDL cholesterol levels (17-21) and adrenal cholesterol stores (18). The finding that hepatic SR-BI overexpression leads to significant increases in biliary cholesterol content (17, 32) is consistent with gene-targeting studies (18,19) that suggest an important role for SR-BI in RCT. In addition to HDL, SR-BI can bind other ligands, including lipoproteins [LDL, modified LDL, very low density lipoprotein (VLDL)] and apolipoproteins (33-37), and can mediate efflux...

Lipid A is the active moiety of lipopolysaccharide (LPS, also referred to as endotoxin), a surface component of Gram-negative bacteria that stimulates macrophage activation and causes endotoxic shock. Macrophages can bind, internalize and partially degrade LPS, lipid A and its bioactive precursor, lipid IVA. We report here that lipid IVA binding and subsequent metabolism to a less active form by macrophage-like RAW 264.7 cells is mediated by the macrophage scavenger receptor. Scavenger-receptor ligands inhibit lipid IVA binding to, and metabolism by, RAW cells, and lipid IVA binds to type I and type II bovine scavenger receptors on transfected Chinese hamster ovary cells. Although in vitro competition studies with RAW cells indicate that scavenger receptor binding is not involved in LPS or lipid IVA-induced stimulation of macrophages, in vivo studies show that scavenger-receptor ligands greatly inhibit hepatic uptake of lipid IVA in mice. Thus, scavenger receptors expressed on macrophages may have an important role in the clearance and detoxification of endotoxin in animals.

The high-density lipoprotein (HDL) receptor, scavenger receptor, class B, type I (SR-BI), mediates both the selective uptake of lipids, mainly cholesterol esters, from HDL to cells and the efflux of cholesterol from cells to lipoproteins. The mechanism underlying these lipid transfers is distinct from classic receptor-mediated endocytosis, but it remains poorly understood. To investigate SR-BI's mechanism of action and in vivo function, we developed a high-throughput screen to identify small molecule inhibitors of SR-BI-mediated lipid transfer in intact cells. We identified five compounds that in the low nanomolar to micromolar range block lipid transport (BLTs), both selective uptake and efflux. The effects of these compounds were highly specific to the SR-BI pathway, because they didn't interfere with receptor-mediated endocytosis or with other forms of intracellular vesicular traffic. Surprisingly, all five BLTs enhanced, rather than inhibited, HDL binding by increasing SR-BI's binding affinity for HDL (decreased dissociation rates). Thus, the BLTs provide strong evidence for a mechanistic coupling between HDL binding and lipid transport and may serve as a starting point for the development of pharmacologically useful modifiers of SR-BI activity and, thus, HDL metabolism.

The macrophage scavenger receptor, which has been implicated in the pathogenesis of atherosclerosis, has an unusually broad binding specificity. Ligands include modified low-density lipoprotein and some polyanions (for example, poly(I) but not poly(C]. The scavenger receptor type I (ref. 3) has three principal extracellular domains that could participate in ligand binding: two fibrous coiled-coil domains (alpha-helical coiled-coil domain IV and collagen-like domain V), and the 110-amino-acid cysteine-rich C-terminal domain VI. We have cloned complementary DNAs encoding a second scavenger receptor which we have termed type II. This receptor is identical to the type I receptor, except that the cysteine-rich domain is replaced by a six-residue C terminus. Despite this truncation, the type II receptor mediates endocytosis of chemically modified low-density lipoprotein with high affinity and specificity, similar to that of the type I receptor. Therefore one or both of the extracellular fibrous domains are responsible for the unusual ligand-binding specificity of the receptor.

Objective-Scavenger receptor class B type I (SR-BI) is a cell-surface HDL receptor that is implicated in reverse cholesterol transport and protection against atherosclerosis. We have previously demonstrated that SR-BI/apolipoprotein E double-knockout mice develop severe occlusive coronary artery disease and myocardial infarction and die at Ϸ6 weeks of age. To determine if this is a general effect of a lack of SR-BI, we generated mice deficient in both SR-BI and the LDL receptor. Methods and Results-Complete ablation of SR-BI expression in LDL receptor knockout mice resulted in increased plasma cholesterol associated with HDL particles of abnormally large size and a 6-fold increase in diet-induced aortic atherosclerosis but no macroscopic evidence of early-onset coronary artery disease, cardiac pathology, or early death. Furthermore, selective elimination of SR-BI expression in bone marrow-derived cells resulted in increased diet-induced atherosclerosis in LDL receptor knockout mice without concomitant alterations in the distributions of plasma lipoprotein cholesterol. 1 The ability of HDL to protect against atherosclerosis 2 may involve several mechanisms, eg, protecting LDL from oxidation and efficient scavenger receptor-mediated uptake 3 and regulating endothelial cell metabolism (eg, controlling endothelial NO synthase activity via scavenger receptor class B type I [SR-BI] 4,5 ). HDL also mediates reverse cholesterol transport (RCT) 6,7 in which cholesterol is transferred from macrophage foam cells to HDL (efflux), esterified, delivered to liver (directly from HDL or indirectly after transfer to other lipoproteins), and subsequently recycled or secreted in bile. Hepatic (and steroidogenic cell) uptake of cholesteryl esters directly from HDL involves selective lipid uptake, the net transfer of mainly neutral lipids of HDL without net uptake and degradation of its apolipoproteins. 8 Conclusions-SR-BI See page 1486SR-BI mediates physiologically relevant selective HDL lipid uptake. 9 -14 Hepatic SR-BI overexpression in mice drastically reduces plasma HDL cholesterol and increases biliary cholesterol levels. 10 -12 Conversely, complete elimination of SR-BI in mice (null mutant) increases plasma HDL cholesterol in abnormally large HDL particles, decreases biliary cholesterol levels, and reduces lipid stores in steroidogenic tissues. 14 -16 Mice with partially reduced SR-BI attributable to a promoter insertion (SR-BIatt mouse) exhibit phenotypes similar to those of heterozygous null mutants (hepatic SR-BI expression Ϸ50% of control 13,14 ). Analysis of these mice confirmed and extended conclusions drawn from the heterozygous and homozygous null SR-BI knockout (KO) mice. 13,14,17,18 SR-BI can also mediate efflux of unesterified cholesterol to HDL in cultured cells 19 ; however, the physiological significance of this activity is unclear.The critical role of SR-BI in HDL metabolism suggested that SR-BI expression levels might influence development of atherosclerosis. We and others have examined the effects of loss o...

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The conserved oligomeric Golgi (COG) complex is an eight-subunit (Cog1-8) peripheral Golgi protein involved in Golgi-associated membrane trafficking and glycoconjugate synthesis. We have analyzed the structure and function of COG using Cog1 or Cog2 null Chinese hamster ovary cell mutants, fibroblasts from a patient with Cog7-deficient congenital disorders of glycosylation, and stable Cog5-deficient HeLa cells generated by RNA interference. Although the dilation of some Golgi cisternae in Cog5 Multisubunit peripheral membrane protein complexes appear to play important roles in facilitating Golgi-associated membrane trafficking and glycoconjugate processing (1-10). One of these is the conserved oligomeric Golgi (COG) 2 complex comprising eight distinct subunits, . The initial identification and characterization of low density lipoprotein receptor-deficient mammalian cell mutants with defects in Cog1 and Cog2 subunits that affect the structure and function of the Golgi apparatus (11-13, 18, 24) were followed by genetic and biochemical analysis in yeast (14 -16, 19 -23, 26, 28 -31) and Drosophila melanogaster (32), as well as purification of the COG complex from bovine brain (17,24).Defects in COG function can cause abnormalities in glycoconjugate synthesis, intracellular protein sorting, protein secretion, and, in some cases, cell growth. In mammalian Cog1 (ldlB cells) or Cog2 (ldlC cells) null Chinese hamster ovary (CHO) cell mutants, multiple Golgi cisternae are dilated (24), and there are pleiotropic defects in a number of medial-and trans-Golgi-associated reactions affecting virtually all N-, O-, and lipid-linked glycoconjugates (12). The pleiotropy and heterogeneity of the glycosylation defects suggested that COG influences the regulation, compartmentalization, or activity of multiple Golgi glycosylation enzymes and/or their substrate transporters without substantially disrupting secretion or endocytosis (12,33). Because the activities of many glycosylation-related proteins depend on their proper intra-Golgi localization and appropriate intralumenal environments (7, 34 -42), it was proposed that COG might play a role directly or indirectly in resident Golgi proteins' transport to, retention at, or retrieval to appropriate sites, or that it might otherwise determine the Golgi's structure and/or lumenal environment (12,43). This proposal was supported by the finding that a subset of Golgi type II membrane proteins, called GEARs, is mislocalized and/or abnormally rapidly degraded in Cog1-and Cog2-deficient CHO mutants (43). As expected from the analysis of the CHO cell mutants, a mutation in a gene encoding one of the human COG subunits (COG7) has been shown to be responsible for a rare form of lethal congenital disorders of glycosylation (CDG) (44). In the mutant fibroblasts from the Cog7-deficient patients, which exhibit global glycosylation defects less severe than those in the Cog1 and Cog2 null CHO cells, trafficking of a Golgi-localized glycosylation enzyme, Gal␤1, 3Gal-NAc ␣2,3-sialyltransferase, is impai...