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

https://doi.org/10.1016/j.abb.2010.02.011Get rights and content

Abstract

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.

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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