Abstract 392: The Role of Scavenger Receptor-BI’s Transmembrane Domains in Cholesterol Transport: A "Locked Dimer" Strategy

2017 ◽  
Vol 37 (suppl_1) ◽  
Author(s):  
Sarah Proudfoot ◽  
Alexandra Chadwick ◽  
Emma Allen ◽  
Daisy Sahoo
Keyword(s):  
Disulfide Bonds ◽  
Free Cholesterol ◽  
Cholesterol Oxidase ◽  
Scavenger Receptor ◽  
Density Lipoprotein ◽  
Immunoblot Analysis ◽  
Surface Expression ◽  
Transmembrane Domains ◽  
Cell Surface Expression ◽  
Cholesterol Transport

Efficient reverse cholesterol transport requires interactions between high density lipoprotein (HDL) and its receptor, scavenger receptor-BI (SR-BI). SR-BI is an 82 kDa protein with a large extracellular domain anchored by two transmembrane domains (TMDs). Our lab recently solved the NMR structure of SR-BI’s C-terminal TMD (C-TMD), a region that mediates SR-BI dimerization. Further, FRET studies suggest HDL-induced movement between neighboring SR-BI monomers, which led to our hypothesis that flexibility between SR-BI TMDs facilitates cholesterol transport. Using structure-guided mutagenesis, we introduced cysteine residues into the C-TMD of full-length SR-BI to create “locked dimers” of the receptor. Total lysate and cell surface expression of WT-, A444C-, L451C-, or G453C-SR-BI were verified in transiently-transfected COS-7 cells by immunoblot analysis and flow cytometry, respectively. Based on the predicted orientation of sulfhydryl side chains relative to the putative dimerization motif, we used immunoblot analysis following electrophoresis under reducing/non-reducing conditions to confirm that A444C- and L451C-SR-BI, but not G453C-SR-BI, formed disulfide bonds. Compared to WT-SR-BI, the locked dimer mutants, A444C- and L451C-SR-BI, exhibited normal selective uptake of [ 3 H]-cholesteryl oleyl ether, despite slightly reduced [ 125 I]-HDL binding. SR-BI-mediated cholesterol efflux to HDL from cells pre-labeled with [ 3 H]-cholesterol was also unaltered by the presence of locked dimers. Finally, we investigated the ability of WT and mutant SR-BI receptors to alter accessibility of membrane free cholesterol to exogenous cholesterol oxidase (as judged by cholestenone levels). L451C- or G453C-SR-BI expression led to reduced cholestenone production compared to WT-SR-BI, suggesting that these mutants may be defective in reorganizing pools of membrane cholesterol. In conclusion, our preliminary data suggest that limiting conformational flexibility between TMDs by forcing locked dimers of SR-BI may not have a major impact on SR-BI-mediated cholesterol transport. However, locked dimers of SR-BI appear to affect the ability of SR-BI to modulate plasma membrane pools of free cholesterol, and this deserves further investigation.

Abstract 522: Scavenger Receptor-BI’s Cholesterol Transport Functions are Dependent on its Extracellular Tryptophan Residues

2015 ◽  
Vol 35 (suppl_1) ◽  
Author(s):  
Rebecca L Holme ◽  
James J Miller ◽  
Daisy Sahoo
Keyword(s):  
Cholesterol Oxidase ◽  
Scavenger Receptor ◽  
Density Lipoprotein ◽  
Surface Expression ◽  
Structural Features ◽  
Cell Surface Expression ◽  
Cholesterol Transport ◽  
Scavenger Receptor Bi ◽  
Atherogenic Particle ◽  
Mutant Receptors

High density lipoprotein (HDL) functions as an anti-atherogenic particle, primarily due to its role in reverse cholesterol transport whereby HDL delivers cholesterol to the liver for excretion upon interaction with its receptor, scavenger receptor BI (SR-BI). The extracellular domain of SR-BI is required for its cholesterol transport functions, yet our understanding of the molecular and structural features of this domain remains limited. We designed experiments to test the hypothesis that one or more of the six highly conserved extracellular tryptophan (Trp; W) residues are critical for mediating receptor function. Towards this end, we created a series of Trp-to-Phe mutant receptors of SR-BI, as well as Trp-free SR-BI and assessed the ability of these mutant receptors to mediate cholesterol transport. Wild-type (WT) or mutant SR-BI receptors were transiently expressed in COS7 cells and proper cell surface expression was confirmed by immunoblotting, confocal microscopy and flow cytometry. Next, we showed that Trp-free- and W415F-SR-BI had a significantly decreased ability to bind HDL (12.7% and 31.3% of WT levels, respectively) and promote selective uptake of HDL-cholesteryl esters (35.2% and 70.1% of WT levels, respectively). Interestingly, only Trp-free-, but not W415F-SR-BI, showed an impaired ability to mediate efflux of free cholesterol (FC) (90.8% decrease vs. WT). Furthermore, both W415F- and Trp-free SR-BI were unable to reorganize plasma membrane pools of FC based on lack of sensitivity of FC to exogenous cholesterol oxidase. We then designed an additional set of mutant SR-BI receptors to determine whether restoration of Trp415 alone (or in combination with other Trp residues) could rescue SR-BI function. Restoration of Trp415 into Trp-free-SR-BI partially rescued cholesterol transport functions. Addition of any of the other 5 extracellular Trp residues was also not sufficient to restore WT cholesterol transport function in combination with Trp415. In summary, loss of all Trp residues in SR-BI impairs its cholesterol transport functions, mostly due to the loss of Trp415. Homology modeling of SR-BI based on the crystal structure of LIMP-2, a member of the same protein family, may help identify the importance of this residue in future studies.

scholarly journals Apolipoprotein A1-Related Proteins and Reverse Cholesterol Transport in Antiatherosclerosis Therapy: Recent Progress and Future Perspectives

2022 ◽  
Vol 2022 ◽  
pp. 1-9
Author(s):  
Xiuting Xu ◽  
Zikai Song ◽  
Bao Mao ◽  
Guoliang Xu
Keyword(s):  
Reverse Cholesterol Transport ◽  
Scavenger Receptor ◽  
Cholesterol Acyltransferase ◽  
Density Lipoprotein ◽  
Coronary Artery Occlusion ◽  
Artery Occlusion ◽  
The Body ◽  
Apolipoprotein A1 ◽  
Cholesterol Transport ◽  
Class B

Hyperlipidemia characterized by abnormal deposition of cholesterol in arteries can cause atherosclerosis and coronary artery occlusion, leading to atherosclerotic coronary heart disease. The body prevents atherosclerosis by reverse cholesterol transport to mobilize and excrete cholesterol and other lipids. Apolipoprotein A1, the major component of high-density lipoprotein, plays a key role in reverse cholesterol transport. Here, we reviewed the role of apolipoprotein A1-targeting molecules in antiatherosclerosis therapy, in particular ATP-binding cassette transporter A1, lecithin-cholesterol acyltransferase, and scavenger receptor class B type 1.

Abstract 404: Altered Interaction of Modified High Density Lipoprotein with Scavenger Receptor BI

2013 ◽  
Vol 33 (suppl_1) ◽  
Author(s):  
Alexandra C Chadwick ◽  
Rebecca L Holme ◽  
Paula-Dene C Nesbeth ◽  
Kirkwood A Pritchard ◽  
Daisy Sahoo
Keyword(s):  
Oxidative Stress ◽  
High Density Lipoprotein ◽  
Scavenger Receptor ◽  
Density Lipoprotein ◽  
High Density ◽  
Cholesterol Transport ◽  
Selective Uptake ◽  
Peripheral Tissues ◽  
Scavenger Receptor Bi ◽  
Hdl Function

High density lipoprotein (HDL) combats atherosclerosis, largely through its role in the reverse cholesterol transport (RCT) pathway where excess cholesterol from peripheral tissues is transported by HDL to the liver for excretion. High HDL-cholesterol (HDL-C) levels have been traditionally linked to a lower risk for cardiovascular disease (CVD). However, recent evidence suggests that HDL “function”, rather than HDL levels, is a better indicator of CVD risk as modifications to HDL under oxidative stress can render the particles “dysfunctional”. Scavenger receptor BI (SR-BI), the HDL receptor, mediates the selective uptake of HDL-cholesteryl ester (CE) into the liver during RCT. We hypothesized that SR-BI would be unable to mediate its cholesterol transport functions in the presence of oxidized or modified HDL due to an inability to engage in productive binding interactions with modified ligands. To test this hypothesis, we assessed HDL binding and selective uptake of HDL-CE in COS7 cells transiently expressing SR-BI using native HDL or HDL modified with: 1) copper (Cu2+), 2) 4-hydroxynonenal (HNE), or 3) acrolein. Our data revealed that, compared to native HDL, SR-BI bound 20-50% less Cu2+-HDL and acrolein-HDL, and mediated 40%-60% less selective uptake of CE from these modified particles, respectively. On the other hand, while SR-BI was able to bind HNE-HDL, it could not efficiently mediate cholesterol uptake (20% less compared to native HDL). Interestingly, our data also revealed that the ability of SR-BI to mediate the release of free cholesterol from COS7 cells did not differ when modified HDL served as acceptor particles, as compared to native HDL. Taken together, only the HDL binding and HDL-CE selective uptake functions of SR-BI are influenced by the type of modification on the HDL particle. These data have significant implications as they suggest that higher levels of plasma HDL-C may, in part, be the result of the inability of SR-BI to recognize and mediate cholesterol removal from HDL particles that have been exposed to oxidative stress. More detailed investigations of the interactions between SR-BI and various populations of oxidized HDL will improve our understanding of the mechanisms that render HDL dysfunctional, and ultimately, atherogenic.

scholarly journals The Low-Density Lipoprotein Class A Module of the Relaxin Receptor (Leucine-Rich Repeat Containing G-Protein Coupled Receptor 7): Its Role in Signaling and Trafficking to the Cell Membrane

Endocrinology ◽  
2007 ◽  
Vol 148 (3) ◽  
pp. 1181-1194 ◽  
Author(s):  
András Kern ◽  
Alexander I. Agoulnik ◽  
Gillian D. Bryant-Greenwood
Keyword(s):  
Cell Surface ◽  
Low Density Lipoprotein ◽  
Density Lipoprotein ◽  
Surface Expression ◽  
Cell Surface Expression ◽  
Lipoprotein Class ◽  
Class A ◽  
Relaxin Family ◽  
G Protein Coupled ◽  
Relaxin Receptor

The relaxin receptor (LGR7, relaxin family peptide receptor 1) is a member of the leucine-rich repeat containing G protein-coupled receptors subgroup C. This and the LGR8 (relaxin family peptide receptor 2) receptor are unique in having a low-density lipoprotein class A (LDL-A) module at their N termini. This study was designed to show the role of the LDL-A in LGR7 expression and function. Point mutants for the conserved cysteines (Cys47 and Cys53) and for calcium binding asparagine (Asp58), a mutant with deleted LDL-A domain and chimeric LGR7 receptor with LGR8 LDL-A all showed no cAMP response to human relaxins H1 or H2. We have shown that their cell surface delivery was uncompromised. The mutation of the putative N-linked glycosylation site (Asn36) decreased cAMP production and reduced cell surface expression to 37% of the wild-type LGR7. All point mutant, chimeric, and wild-type receptor proteins were expressed as the two forms. The immature or precursor form of the receptor was 80 kDa, whereas the mature receptor, delivered to the cell surface was 95 kDa. The glycosylation mutant was also expressed as two forms with appropriately smaller molecular masses. Deletion of the LDL-A module resulted in expression of the mature receptor only. These data suggest that the LDL-A module of LGR7 influences receptor maturation, cell surface expression, and relaxin-activated signal transduction.

Abnormal intracellular sorting of O-linked carbohydrate-deficient interleukin-2 receptors

1988 ◽  
Vol 8 (8) ◽  
pp. 3357-3363
Author(s):  
K F Kozarsky ◽  
S M Call ◽  
S K Dower ◽  
M Krieger
Keyword(s):  
Cell Surface ◽  
Cho Cells ◽  
Interleukin 2 ◽  
Chinese Hamster ◽  
Density Lipoprotein ◽  
Surface Expression ◽  
Cell Surface Expression ◽  
Structure Stability ◽  
Wild Type ◽  
Intracellular Sorting

The synthesis and intracellular sorting of the interleukin-2 (IL-2) receptor were studied with a line of mutant Chinese hamster ovary (CHO) cells with a reversible defect in protein O glycosylation. Under normal culture conditions the mutant ldlD cannot add N-acetylgalactosamine (Ga1NAc) to proteins. Ga1NAc is the first sugar of mucin-type O-linked oligosaccharides attached to protein. This O-glycosylation defect is rapidly corrected when Ga1NAc is added to the culture mediu. An expression vector for the p55 human IL-2 receptor was transfected into wild-type CHO and ldlD cells and the structure, stability, and cell surface expression of the receptor were examined by immunoprecipitation and antibody-binding assays. Essentially all of the mature form of the normally glycosylated IL-2 receptor in both wild-type CHO cells and ldlD cells incubated with Ga1NAc was expressed on the cell surface. The stability of O-linked carbohydrate-deficient (Od) IL-2 receptors (in ldlD cells without Ga1NAc) was normal; however, missorting of the Od receptors resulted in very little cell surface expression. The sialidase sensitivity and endoglycosidase H resistance of mature Od IL-2 receptors suggest that Od receptor missorting occurred in or beyond the trans Golgi apparatus. The abnormal sorting of the Od IL-2 receptor is compared with the O-glycosylation dependence of the surface expression and stability of the low-density lipoprotein receptor, decay-accelerating factor, and the major antigen envelope protein of Epstein-Barr virus.

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