Effects of amino acid substitutions at glycine 420 on SR-BI cholesterol transport function

Parathath, Saj; Darlington, Yolanda F.; Moya, Margarita de la Llera; Drazul-Schrader, Denise; Williams, David L.; Phillips, Michael C.; Rothblat, George H.; Connelly, Margery A.

doi:10.1194/jlr.m700086-jlr200

Cited by 7 publications

(10 citation statements)

References 77 publications

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“…We examined the functional importance of each individual leucine within this motif of SR-BI, and used NMR analysis to further understand why several single leucine mutations lead to disruption of different receptor functions. We observed that mutation of L413 significantly disrupts HDL binding, HDL-CE uptake, and cholestenone production and corroborates previous findings (Parathath et al, 2007; Parathath et al, 2004; Sahoo et al, 2007b) that residues in this region are imperative for all of these cholesterol transport functions. Indeed, we recently showed that mutation of tryptophan 415 to phenylalanine disrupted several SR-BI functions (Holme et al, 2016).…”

Section: Discussionsupporting

confidence: 91%

NMR Structure of the C-Terminal Transmembrane Domain of the HDL Receptor, SR-BI, and a Functionally Relevant Leucine Zipper Motif

et al. 2017

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SUMMARY The interaction of high density lipoprotein (HDL) with its receptor, scavenger receptor BI (SR-BI), is critical for lowering plasma cholesterol levels and reducing the risk for cardiovascular disease. The HDL/SR-BI complex facilitates delivery of cholesterol into cells and is likely mediated by receptor dimerization. This work describes the use of nuclear magnetic resonance (NMR) spectroscopy to generate the first high-resolution structure of the C-terminal transmembrane domain of SR-BI. This region of SR-BI harbors a leucine zipper dimerization motif, that when mutated, impairs the ability of the receptor to bind HDL and mediate cholesterol delivery. These losses in function correlate with the inability of SR-BI to form dimers. We also identify juxtamembrane regions of the extracellular domain of SR-BI that may interact with the lipid surface to facilitate cholesterol transport functions of the receptor.

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Section: Discussionsupporting

confidence: 91%

NMR Structure of the C-Terminal Transmembrane Domain of the HDL Receptor, SR-BI, and a Functionally Relevant Leucine Zipper Motif

et al. 2017

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“…It is possible that the levels of selective uptake efficiency were artificially high as a result of the low amount of HDL binding. This trend has been observed in a previous study 38 . It is also possible that mutation of Trp 415 to Phe results in a conformational change within the receptor that makes it less favorable for HDL binding.…”

Section: Discussionsupporting

confidence: 90%

Tryptophan 415 Is Critical for the Cholesterol Transport Functions of Scavenger Receptor BI

et al. 2015

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High density lipoproteins (HDL) are anti-atherogenic particles, primarily due to their role in the reverse cholesterol transport pathway whereby HDL delivers cholesteryl esters (CE) to the liver for excretion upon interaction with its receptor, scavenger receptor BI (SR-BI). We designed experiments to test the hypothesis that one or more of the eight highly conserved tryptophan (Trp; W) residues in SR-BI are critical for mediating function. We created a series of Trp-to-phenylalanine (Phe, F) mutant receptors, as well as Trp-less SR-BI (ΔW-SR-BI), and assessed their ability to mediate cholesterol transport. Wild-type (WT) or mutant SR-BI receptors were transiently expressed in COS-7 cells, and cell surface expression was confirmed. Next, we showed that Trp-less- and W415F-SR-BI had significantly decreased abilities to bind HDL and promote selective uptake of HDL-CE, albeit with higher selective uptake efficiency as compared to WT-SR-BI. Interestingly, only Trp-less-, but not W415F-SR-BI, showed an impaired ability to mediate efflux of free cholesterol (FC). Furthermore, both W415F- and Trp-less-SR-BI were unable to reorganize plasma membrane pools of FC based on lack of sensitivity to exogenous cholesterol oxidase. Restoration of Trp 415 into the Trp-less-SR-BI background was unable to rescue Trp-less-SR-BI’s impaired functions, suggesting that Trp 415 is critical, but not sufficient for full receptor function. Furthermore, with the exception of Trp 262, restoration of individual extracellular Trp residues, in combination with Trp 415, into the Trp-less-SR-BI background partially rescued SR-BI function, indicating that Trp 415 must be present in combination with other Trp residues for proper cholesterol transport functions.

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“…The C251S, C323S, C384S, and Cys-less mutant receptors were produced and sequenced by TOP Gene Technologies (Pointe-Claire, Quebec, Canada). Myc-SR-BI (36) and G420C-SR-BI (40) were previously described.…”

Section: Methodsmentioning

confidence: 99%

Extracellular Disulfide Bonds Support Scavenger Receptor Class B Type I-Mediated Cholesterol Transport

2011

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Scavenger receptor class B, type I (SR-BI) binds HDL and mediates the selective uptake of cholesteryl esters (CE). Although the extracellular domain of SR-BI is critical for function, the structural characteristics of this region remain elusive. Using sulfhydryl-labeling strategies, we report the novel finding that all six cysteine (Cys) residues in the extracellular domain of SR-BI are involved in disulfide bond formation that is intramolecular by nature. We hypothesize that an SR-BI conformation stabilized by extracellular disulfide bonds is a prerequisite of SR-BI-mediated cholesterol transport. Thus, single Cys mutant SR-BI receptors (C251S-, C280S-, C321S-, C323S-, C334S- and C384S-SR-BI), as well as Cys-less-SR-BI, a mutant SR-BI receptor void of all Cys residues, were created and plasma membrane localization was confirmed. Functional assays revealed that C280S-, C321S-, C323S-, C334S- and Cys-less SR-BI mutant receptors displayed reduced HDL binding and subsequent selective uptake of HDL-CE. However, only C323S-SR-BI and Cys-less-SR-BI were unable to mediate wild-type levels of efflux of free cholesterol (FC) to HDL. None of the Cys mutations disrupted SR-BI’s ability to redistribute plasma membrane FC. Taken together, the intramolecular disulfide bonds in the extracellular domain of SR-BI appear to maintain the receptor in a conformation integral to its cholesterol transport functions.

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Effects of amino acid substitutions at glycine 420 on SR-BI cholesterol transport function

Cited by 7 publications

References 77 publications

NMR Structure of the C-Terminal Transmembrane Domain of the HDL Receptor, SR-BI, and a Functionally Relevant Leucine Zipper Motif

NMR Structure of the C-Terminal Transmembrane Domain of the HDL Receptor, SR-BI, and a Functionally Relevant Leucine Zipper Motif

Tryptophan 415 Is Critical for the Cholesterol Transport Functions of Scavenger Receptor BI

Extracellular Disulfide Bonds Support Scavenger Receptor Class B Type I-Mediated Cholesterol Transport

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