Thematic Review Series: Lipid Transfer Proteins Scavenger receptor B type 1: expression, molecular regulation, and cholesterol transport function

The SR-B1 gene (Scarb1) was almost simultaneously cloned as a cluster determinant 36 (CD36)-related class B scavenger receptor by expression cloning using cDNA libraries from a Chinese hamster ovary (CHO) cell line, Var-261, with a mutated LDL receptor gene (), and as CD36 and lysosomal integral membrane protein (LIMP) II analogous-I (CLA-1) from human erythroleukemia (HEL) cells based on its sequence similarity to CD36 and LIMP-2 (). CLA-1 is a human homolog of SR-B1. Subsequently, the isolation and characterization of SR-B1 from mouse (), rat (), rabbit (), pig (), cow (), and Northern tree shrew () have been described. Further studies by Monty Krieger's group revealed that SR-B1 binds HDL and mediates the selective delivery of HDL-CE (). This led to identification of SR-B1 as the first bona fide HDL receptor ().

SR-B1 is a member of the class B family of scavenger receptors, which is also known as the CD36 superfamily of scavenger receptors, which are plasma membrane receptors that recognize and take up macromolecules that have a negative charge. Although classified as a group, the scavenger receptor family members are structurally heterogeneous; specifically, the scavenger receptor B family members have a very different molecular structure from other scavenger receptors by having two transmembrane domains with both the N and C termini of the proteins residing intracellularly (). Proteins in this group are cell surface-associated glycoproteins, which include SR-B1 and its splicing variant, SR-B2 (SR-BII), its human homolog, CLA-1/human SR-B1, and spliced variant, CLA-2/human SR-B2, CD36, and LIMP-2 (, , , , , , ). Full-length SR-B1 encodes a 509 amino acid protein, which, like other family members, contains a short N-terminal cytoplasmic domain of 9 amino acids (N-terminal domain), a transmembrane spanning domain of 22 amino acids, a large extracellular domain of 408 amino acids containing a cysteine-rich region and multiple sites for N-linked glycosylation, a second transmembrane domain of 23 amino acids, and a cytoplasmic C-terminal domain of 44 amino acids (, ). SR-B1 is posttranslationally glycosylated, and mutational analysis showed the importance of Asn-108 and Asn-173 for plasma membrane localization of SR-B1 and for the ability to transfer lipid from HDL to cells (). The mouse SR-B1 gene is located on chromosome 5 with 13 exons and 12 introns. Due to alternative splicing, the second isoform of SR-B1, SRB2 (SR-BII), has only 12 exons and a different C terminal with 41 amino acids (). Using a specific antibody for SR-B2 isoform, SR-B2 protein was detected in mouse liver, testis, and adrenal glands at about 5–12% of immunodetectable SR-B1 (, ). Recent genomic analysis revealed a third SR-B1 variant with another different alternative splicing that results in 11 exons and 520 amino acids; however, no functional significance of this variant has been reported yet. The mouse SR-B1 gene exon organization and the C termini of the three variants, as well as a cartoon of the structural features of SR-B1, are shown in Fig. 1. The predicted molecular mass of SR-B1 is ∼57 kDa, but, because the protein is heavily glycosylated (), it runs ∼83–84 kDa on Western blot (, ). SR-B1 is also myristoylated and palmitoylated, but fatty acid acylation of the protein does not affect its selective HDL-CE transport function ().

Scavenger receptors are cell surface membrane proteins that can bind chemically modified lipoproteins, including acetylated LDL (AcLDL), oxidized LDL (OxLDL), and often many other types of ligands (, ). SR-B1 was first identified in scavenger receptor expression cloning experiments using AcLDL as a ligand. Further studies have demonstrated that SR-B1 or its human homolog, CLA-1, bind an array of ligands (Table 1), including both native (VLDL, LDL, and HDL) (, ) and modified lipoproteins (OxLDL and AcLDL) (, ). SR-B1 can also bind maleylated BSA (); advanced glycation end-product modified proteins, such as advanced glycation end product BSA (); hypochlorite-modified LDL (, ); discoidal reconstituted phospholipid/unesterified cholesterol particles containing apoA-I, apoA-II, apoC-III, or apoE (, ); reconstituted HDL particles containing apoA-I or apoA-II (); and lipid-free apoE (). However, SR-B1 exhibits much higher affinity for native HDL (, ). The facts that HDL consists of particles of varying sizes and different lipid and protein compositions and densities (), and that apoA-I is the principal component of HDL, are in accordance with observations that the physical characteristics of HDL as well as the conformation/organization of apoA-I in HDL particles are critical for optimal binding of HDL to SR-B1 (). Indeed, it has been demonstrated that spherical α-HDL particles, which are larger in size, cholesterol rich, and lower density, bind more strongly to SR-B1 as compared with high-density lipid-poor preβ-HDL (, ). Interestingly, reconstituted discoidal apoA-I complexes bind SR-B1 with even much greater affinity than native α-HDL (). Furthermore, amphipathic helices of apoA-I have been demonstrated to be critical for its binding to SR-B1 (). The relative levels of apoA-I and apoA-II in spherical α-HDL particles also dictate the association with SR-B1 (, , ). SR-B1 can also bind anionic phospholipids (, , ), serum amyloid A (), silica (), oxidized phospholipids (, ), and bacterial cell-wall components, such as endotoxin [lipopolysaccharides (LPSs)] of gram-negative bacteria and lipoteichoic acid of gram-positive bacteria (, ). Additionally, SR-B1/CLA-1 can bind and internalize other bacterial and mammalian proteins containing amphipathic helices (, , ). Finally, SR-B1 has been shown to bind several types of apoptotic cells (, , , , , ). From the perspective of displaying a broad spectrum of ligand recognition, the most closely related proteins are from the LDL receptor family (, ). Interestingly, the structure of the SR-B1 family is distinct from that of the LDL receptor family proteins, again by having two transmembrane domains with both the N and C termini of the protein residing intracellularly, while LDL receptor family members have a single transmembrane domain.

SR-B1 is expressed in a wide variety of tissues and cell types, including adipocytes, macrophages, endothelial cells, intestine, keratinocytes, epithelial cells, smooth muscle cells, monocytes, placenta, gallbladder, and ocular tissues (, , , , , ). However, in rodents, SR-B1 is expressed most abundantly in the liver and steroidogenic cells of the adrenal gland, testis, and ovary (), tissues that efficiently utilize the selective pathway in deriving CEs for use in bile acid synthesis and biliary cholesterol secretion (, , , ) or steroid hormone production (, , , , , , ). In humans, CLA-1/human SR-B1 shows a similar tissue distribution (, ). Interestingly, variable expression levels of SR-B2 mRNA are also detected in mouse liver, adrenal gland, and testis, with SR-B2 transcripts ranging from 13% to 40% of total SR-B1 in mouse liver (, ), from 6% to 30% in adrenal (, ), and 80% in whole testis (). Moreover, of the total hepatic SR-B1/SR-B2 mRNA, only 12% of SR-B1/SR-B2 protein is detected in mouse liver by Western blotting, possibly because of less efficient translation of the SR-B2 protein ().

It is of interest that steroidogenic tissues, which express high levels of SR-B1 in vivo, are equipped with an intricate microvillar system for the trapping of lipoproteins (, , , , , ). This general region of steroidogenic cells is referred to as the microvillar compartment, and the specialized spaces created between adjacent microvilli are called microvillar channels. It is the microvillar channels where the various lipoproteins are trapped prior to the selective uptake of lipoprotein-CEs into cells. Use of electron microscopic immunocytochemistry techniques revealed heavy labeling of SR-B1 specifically in these regions (corresponding to microvilli and microvillar channels), and current evidence suggests that these microvillar compartments express high levels of SR-B1 to facilitate the selective uptake of HDL-CE in steroidogenic tissues. Figure 2 displays photomicrographs demonstrating the microvillar system. In this context, we previously demonstrated that expression of SR-B1 in heterologous (Sf9 insect cells) () or homologous (Y1-BS1 mouse adrenocortical cells) () cell systems promotes microvillar channel formation and selective HDL-CE uptake. Furthermore, examination of SR-B1 knockout mice provided further evidence that SR-B1 is required for formation of microvilli and microvillar channels. Unlike their wild-type counterparts, the adrenal zona fasciculata cells from SR-B1-null mice exhibit disorganized microvilli, devoid of microvillar channels with an absence of HDL particles ().

SR-B1 expression has also been reported in several nonmammalian vertebrates, as well as in several insect species. High expression of SR-B1 is reported in the liver, ovary, heart, and blood vessels in the turtle and in the liver of chickens, frogs, goldfish, sharks, and skates (). SR-B1 expression is reported in the liver of Atlantic salmon (Salmo salar L.) () and in several organs of prawns (Macrobrachium nipponense) (). Twelve to fourteen candidate SNMP/CD36 homologs from each of the genomes of Drosophila melanogaster, Drosophila pseudoobscura, Anopheles gambiae, and Aedes aegypti (Diptera), eight candidate homologs from Apis mellifera (Hymenoptera), and 15 from Tribolium castaneum (Coleoptera) have been identified (, ). The 14 D. melanogaster SNMP/CD36 homologs include CG10345, CG1887, CG2736, CG31741, CG3829, CG40006, CG7227, CG7422, crq, emp, ninaD, pes, santa-maria, and Snmp (). In the silkworm, two genes, Cameo1 and Cameo2, encode proteins homologous to SR-B1 (, ). There are 13 other genes homologous to Cameo1 and Cameo2. These genes are SCRB5, SCRB6, SCRB7, SCRB8, SCRB9, SCRB10, SCRB11, SCRB12, SCRB13, SCRB14, SCRB15, SNMP1, and SNMP2 ().

Various approaches, including site-directed mutagenesis, “domain swap,” and chemical cross-linking, have been utilized and have identified several structural features to be important for the function of SR-B1 (Fig. 1C). The C-terminal intracellular domain contains an interacting domain (VLQEAKL) for binding with PDZ domain-containing protein, through which the function of SR-B1 is regulated in a tissue-specific manner (for detail see below). SR-B1 was shown to form oligomers, which display improved function (, , , , , ), with several portions involved in oligomerization. An N-terminal transmembrane glycine motif (G15_G18_G25) was shown to be required for oligomerization and lipid transport (), whereas studies using fluorescence resonance energy transfer techniques to visualize SR-B1 homo-oligomerization revealed that the C termini of SR-B1 appeared to be involved in its homo-dimerization (). Meanwhile, using a sulfhydryl-reactive reagent, the six conserved cysteine residues (C251, C280, C321, C323, C334, and C384) found in the ectodomain of SR-B1 were also demonstrated to be involved in dimer/oligomer formation (). Experiments using chimeric SR-B1 and CD36 revealed that the N terminal of the extracellular domain is important for unesterified cholesterol efflux to HDL (, ).

Several recent studies have also examined the impact of cysteine residues on cellular trafficking and selective HDL-CE transport function (, , , , ). These studies were achieved primarily by mutating cysteine residues in mouse, rat, or human SR-B1 individually or in certain combinations to either Ser or Gly, and subsequent expression of individual constructs in cultured cells followed by determination of receptor activity, subcellular distribution of receptor protein, or selective lipid transport function. Two reports demonstrated that four cysteine residues, C280, C321, C323, and C334, may be involved in disulfide bond formation and critically involved in the expression and function of SR-B1 (, ). In addition, Yu et al. (), using a mass spectrometry technique, have reported the identification of two disulfide bonds in SR-B1 that connect cysteine residues within the conserved C321-P322-C323 (CPC) motif and connect C280 to C334 and a reduced cysteine side chain that contribute to the functional expression of SR-B1. Using a C323G mutant, a blocking antibody against C323 region, and a C323G mutant transgenic mouse model, Guo et al. () demonstrated that C323 plays a critical role in HDL binding to cell surface SR-B1 and SR-B1-mediated selective HDL-CE uptake. An exoplasmic cysteine, C384, of SR-B1 has also been implicated in SR-B1-mediated selective HDL-CE transport activity ().

The crystal structure of LIMP-2 was obtained recently and showed that the main ectodomain of the protein contains an antiparallel β-barrel core with many short α-helical segments (). Two disulphide bridges stabilize the fold. The disulphide bridge pattern for LIMP-2 (C274-C329 and C312-C318) is similar to that predicted for SR-B1 (C321-C323, C274-C329) and for CD36 (C313-C322, C272-C333), and is consistent with experimental data (, , ). Most of the proteins in this family have lipid transport activity, and the crystal structure of LIMP-2 showed that eight amino acids work coordinately to form a tunnel cavity that spans the entire length of the ectodomain, allowing facilitated lipid transfer. These eight acidic and basic amino acids form a network of hydrogen and ionic bonds and contribute to the lining of the cavity, which is predominately hydrophobic to accommodate lipid moieties. Figure 4 displays the structure of SR-B1, both as a monomer and as a dimer, modeled on the crystal structure of LIMP-2, with the eight amino acids comprising the tunnel cavity highlighted. As illustrated, there are conformational changes in the tunnel cavity that are predicted to occur during the transition between monomer and dimer. Further experimental studies will be needed to explore the functional significance of the structural transition between monomer to dimer.

Recently, a fragment of SR-B1 (residues 405-475), which includes the C-terminal transmembrane domain, was purified and reconstituted in detergent micelles (). Analysis of this fragment using NMR has generated high-resolution structural information of the C-terminal transmembrane domain of SR-B1 (), which revealed that there are two short α-helices (residues 409-419 and 427-436) and a long α-helix spanning the entire transmembrane domain (residues 438-469). There is a potential GXXXG glycine dimerization motif starting at G420 together with a putative leucine zipper motif spanning from amino acid 413 to amino acid 455. Mutational analysis showed that mutants L413A and L448A resulted in decreased receptor efflux of unesterified cholesterol to HDL. G420 was previously shown to be important for SR-B1 to function for CE delivery ().When a smaller SR-B1 fragment containing residues 405-445, which lacks the hydrophobic transmembrane domain, was purified and examined by NMR, this fragment was shown to require a hydrophobic environment to fold properly, suggesting the existence of a potential membrane-interacting juxtamembrane domain.

As mentioned above, oligomerization of SR-B1 improves its function. Recently a crystal structure of LIMP-2 dimer bound with cholesterol and phosphatidylcholine was obtained. Further analysis revealed that LIMP-2 switches from a monomeric form to a dimeric form when binding to different ligands. When a monomer, it binds glucocerebrosidase; whereas as a dimer, it preferentially binds anionic phosphatidylserine (). This shifting of ligand specificity through oligomerization could be a mechanism of regulation shared with other proteins in the family, such as SR-B1 and CD36, but awaits experimental evidence.

A major function of SR-B1 is to facilitate the transport of CEs from HDL or other lipoproteins to cells by a nonendocytic mechanism referred to as selective uptake (, , , , , , , ). Numerous studies have demonstrated that SR-B1-mediated selective uptake of CE does not show a specificity toward a single class of lipoproteins. It can utilize a number of lipoproteins, including human and rat HDL, reconstituted synthetic HDL particles containing apoA-I, apoC, or native or modified apoE, IDL, and LDL as donors of CE for SR-B1-mediated selective CE uptake in vitro and/or in vivo (, ). However, because these donor lipoproteins differ in characteristics, such as apolipoprotein conformation and apolipoprotein and lipid composition, as well as the fact that some of these lipoproteins may serve as ligands for other important pathways (e.g., human LDL and apoE-rich rodent HDL are potent ligands for the LDL receptor endocytic pathway), these lipoproteins do not deliver CEs to cells with similar efficacies. Based on the evidence obtained using CD36/SR-B1 chimeras (, ), SR-B1 mutant (N173Q) (), apoA-I mutants (), apoA-I^−/− HDL particles (, ), and a chemical inhibitor of selective CE transport (), it appears that initial binding of HDL to cell surface-associated SR-B1 and subsequent selective CE transport to cells are independent processes (). The selective uptake process itself can be broadly divided into three steps: 1) binding of cholesterol-rich donor lipoprotein particles to the loop domain of SR-B1; 2) SR-B1-mediated transfer of CEs from the lipoprotein particles to the plasma membrane; and 3) the release of the cholesterol-poor lipoprotein particles back into the circulation (, , ). Among these steps, the mechanism by which SR-B1 facilitates the transfer of CEs to the plasma membrane is not fully understood. Rodrigueza et al. () provided evidence in favor of a model in which SR-B1 forms a nonaqueous channel between lipoprotein particles and the cell membrane through which CEs move down in a concentration gradient manner. The validity of this model is supported by the recent report of the high-resolution crystal structure of the extracellular domain of the lysosomal LIMP-2, and by modeling the structure of SR-B1 and its other family member, CD36 (, ). Importantly, the crystal structure shows the existence of a large cavity or tunnel that traverses the entire length of the molecule. The dimensions (with 5 × 5 Å opening and 22 × 11 × 8 Å cavity) of this hydrophobic tunnel are sufficient to accommodate CE and free cholesterol (FC) molecules, and SR-B1 mutagenesis studies provided direct support for the involvement of this hydrophobic tunnel as a facilitator of cholesterol ester transfer from bound donor HDL particles to the cell surface plasma membrane (, , ). This is further supported by the observation that an inhibitor of selective HDL-CE uptake binds covalently to Cys384, which is located in the lumen of the tunnel. It is predicted that binding of the inhibitor to this site would block cholesterol (ester) transport (, ). Once selectively delivered to the cell, the intact CEs are either transported to lipid droplets for storage () or hydrolyzed extra-lysosomally by neutral cholesteryl esterase (, ) (, , , ) to provide cholesterol substrate for product formation or reesterification. Previously, we provided evidence that hormone-sensitive lipase serves as the neutral cholesterol esterase in adrenal cells and participates in CE turnover and steroidogenesis (, ).

Besides CEs, SR-B1 also mediates the selective uptake of other lipid components of receptor bound HDL particles, including FC, triglycerides (TGs), phospholipids, and α-tocopherol (, , ). Nonpolar FC, CE, and TG are transported five to ten times more efficiently than more polar phospholipid molecules. The relative selective uptake rate constants for CE, FC, TG, and phospholipid (phosphatidylcholine) have been calculated to be 1.0, 1.6, 0.7, and 0.2, respectively (). SR-B1 also promotes the bidirectional flux of FC between cells and lipoproteins (, , , , ). In addition, in the liver, SR-B1 functions in reverse cholesterol transport (, , ), including selective uptake of CEs from HDL into the liver (one of the later steps in reverse cholesterol transport) and biliary cholesterol secretion (the last step in reverse cholesterol transport), thus promoting conversion of hepatic HDL cholesterol (HDL-C) to bile acids (, , ). SR-B1 also alters the composition of lipid domains of plasma membranes, which then leads to changes in FC flux, changes in membrane cholesterol content, or altered physical/chemical properties of membranes (, ).