Extracellular hydrophobic regions in scavenger receptor BI play a key role in mediating HDL-cholesterol transport
Introduction
The inverse correlation between the risk for developing coronary artery disease and plasma concentrations of high density lipoprotein (HDL)1[1], [2] has been attributed to the strong athero-protective effects of HDL that include inhibition of low density lipoprotein oxidation [3], [4] and oxidative damage [5], promotion of endothelial nitric oxide production [6], [7] and vascular reactivity and integrity [8], inhibition of platelet aggregation and coagulation [9], [10] and prevention of thrombosis [11]. However, the primary athero-protective role of HDL stems from its ability to promote the disposal of peripheral cholesterol at the liver via a process termed reverse cholesterol transport [12].
The final step of reverse cholesterol transport involves the movement of cholesterol from HDL to the liver for catabolism. The selective transfer of cholesteryl ester (CE) from HDL to cells is mediated by scavenger receptor class B type I (SR-BI) [13], an 82-kDa glycosylated cell surface receptor [14] highly expressed in the liver and steroidogenic tissues [15], [16], [17]. SR-BI (509 amino acids) consists of a large extracellular domain (403 amino acids) anchored by two transmembrane domains and two short cytoplasmic tails [18]. Transgenic overexpression [19], [20], [21] or hepatic adenoviral infection [22], [23] of SR-BI decreased HDL plasma cholesterol levels and increased cholesterol catabolism and excretion. On the other hand, a 50% reduction in SR-BI expression [17] or full disruption of the SR-BI gene in mice increased plasma HDL-cholesterol levels and reduced neutral lipid stores in the adrenal gland and ovary [24], [25]. Thus, SR-BI is the most physiologically relevant HDL receptor.
SR-BI-mediated selective uptake of HDL-CE is a two-step process: (i) HDL must bind to the extracellular domain of SR-BI and (ii) CE is transferred from HDL to the plasma membrane by a non-endocytic mechanism, without holoparticle uptake or degradation of apolipoproteins [26], [27], [28]. The critical nature of the extracellular domain of SR-BI in CE transfer has been demonstrated through the use of chimeric receptors [29], [30], [31] and insertion of epitope tags into various regions of the extracellular domain of SR-BI [32]. Moreover, antibodies to the extracellular domain blocked HDL-CE-selective uptake and the delivery of HDL-CE to the steroidogenic pathway in cultured adrenocortical cells [33]. In fact, a set of distinct SR-BI-mediated activities appears to be inherent to the extracellular domain, including free cholesterol (FC) efflux and influx, as well as the ability to increase cellular FC mass and enhance sensitivity of membrane FC to exogenous cholesterol oxidase [34].
Our detailed analyses also reveal the presence of evolutionarily conserved sequences with high hydrophobicity within the extracellular domain of SR-BI. We hypothesized that these hydrophobic regions may play a role in mediating the cholesterol transport functions of SR-BI. To test this hypothesis, we used site-directed mutagenesis to generate point mutations that would reduce overall hydrophobicity of the particular regions: V67N, L140Q/L142Q, V164N, V221N, L359Q, and L411Q. We then correlated the changes in hydrophobicity to the effects on HDL binding, selective uptake of HDL-CE and other functions of SR-BI. In addition, we created a second set of point mutations that maintained the overall hydrophobicity of the selected regions (V67L, L140V/L142V, V164L, V221L, L359V, and L411V) to test whether the changes in SR-BI function were due to changes in hydrophobicity or changes in amino acid identity.
Section snippets
Materials
The following antibodies were used: polyclonal anti-SR-BI specific for the C-terminal or the extracellular domain (Novus Biologicals, Inc., Littleton, CO); anti-glyceraldehyde-3-phosphate dehydrogenase (GAPDH; Millipore, Billerica, MA); peroxidase-conjugated goat anti-rabbit secondary IgG (Jackson ImmunoResearch Laboratories, West Grove, PA). Human HDL (1.063–1.21 g/mL) was purchased from Biomedical Technologies, Inc. [125I]Iodine was from Perkin-Elmer, while [3H]cholesterol and [3H]cholesteryl
Rationale for mutagenesis
The traditional depiction of SR-BI with a “horseshoe-like” topology is based upon hydropathy analysis [38] of the predicted amino acid sequence and reveals two membrane-spanning regions. These N- and C-terminal domains display a hydrophobicity index much greater than the typical threshold value and are long enough to span the membrane bilayer [39], [40]. However, the hydropathy plot (Fig. 1) also revealed that the extracellular domain of SR-BI contains other evolutionarily conserved sequences
Discussion
Since the identification of SR-BI as the HDL receptor [15], [52], numerous studies have examined the mechanism of the HDL-CE selective uptake process via SR-BI. However, few of these reports have commented on how the topology and/or structural organization of SR-BI at the plasma membrane may influence this crucial step in reverse cholesterol transport. We investigated the role of several putative hydrophobic regions of the extracellular domain of SR-BI by creating amino acid substitutions that
Acknowledgments
The authors thank Rhiannon Ledgerwood for excellent technical assistance. This study was supported by National Institutes of Health Grant HL-58012 (to D.S.).
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