Prostaglandin F2α via Yin Yang-1 Suppresses Steroidogenic Acute Regulatory Protein Intracellular Cholesterol Transport while Maintaining Scavenger Receptor Class B Type I Cell Surface Expression in Luteal Cells.

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.

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

Biochemistry ◽

10.1021/bi2005625 ◽

2011 ◽

Vol 50 (28) ◽

pp. 6245-6254 ◽

Cited By ~ 17

Author(s):

Gabriella A. Papale ◽

Paul J. Hanson ◽

Daisy Sahoo

Keyword(s):

Disulfide Bonds ◽

Scavenger Receptor ◽

Cholesterol Transport ◽

Type I ◽

Scavenger Receptor Class ◽

Class B

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Probucol increases hepatic expression of High Density Lipoprotein (HDL) receptor, scavenger receptor class B type I (SR-BI), in vitro and in vivo. — enhancement of reverse cholesterol transport (RCT)

Atherosclerosis ◽

10.1016/s0021-9150(00)80417-7 ◽

2000 ◽

Vol 151 (1) ◽

pp. 92

Author(s):

K. Tsujii ◽

K. Hirano ◽

F. Matsuura ◽

Y. Nakagawa-Toyama ◽

T. Sugimoto ◽

...

Keyword(s):

Reverse Cholesterol Transport ◽

Scavenger Receptor ◽

Density Lipoprotein ◽

Cholesterol Transport ◽

Type I ◽

Hepatic Expression ◽

Scavenger Receptor Class ◽

Class B

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Deficiency of Scavenger Receptor Class B Type I Negatively Affects Progesterone Secretion in Human Granulosa Cells

Endocrinology ◽

10.1210/en.2010-0347 ◽

2010 ◽

Vol 151 (11) ◽

pp. 5519-5527 ◽

Cited By ~ 14

Author(s):

Antonina Kolmakova ◽

Jiangxia Wang ◽

Rebecca Brogan ◽

Charles Chaffin ◽

Annabelle Rodriguez

Keyword(s):

Granulosa Cells ◽

Regulatory Protein ◽

Scavenger Receptor ◽

Ldl Receptor ◽

Density Lipoprotein ◽

Type I ◽

Human Granulosa Cells ◽

Scavenger Receptor Class ◽

Progesterone Secretion ◽

Class B

Our goal was to examine the effect of deficiency of the lipoprotein receptor, scavenger receptor class B type I (SR-BI), on progesterone secretion in human granulosa cells (HGL5). Scrambled or SR-BI small interfering RNA [knockdown (KD)] cells were exposed to dimethylsulfoxide [DMSO, vehicle for forskolin (Fo)], Fo, serum, high-density lipoprotein, low-density lipoprotein (LDL), or Fo plus lipoproteins or serum for 24 h. Progesterone secretion was lower in all of the SR-BI KD cells regardless of treatment. We examined progesterone secretion in SR-BI KD, LDL receptor KD, and double KD cells incubated with DMSO, Fo, LDL, or Fo + LDL for 6–24 h. As compared with scrambled cells, progesterone secretion was lower in SR-BI and double KD cells regardless of treatment; whereas progesterone secretion was only lower in LDL receptor KD cells incubated with LDL and Fo + LDL. We measured phosphorylation of hormone-sensitive lipase (pHSL) expression, intracellular total cholesterol (TC) mass, and progesterone secretion in scrambled and SR-BI KD cells incubated with DMSO or Fo for 2–24 h. The expression of pHSL was similar between the cells and conditions. The mean change in TC mass and progesterone secretion was lower in SR-BI KD cells exposed to DMSO and Fo. Incubating SR-BI KD cells with 22-hydroxy cholesterol did not overcome the reduction in progesterone secretion. At different time points, RNA expression of steroidogenic acute regulatory protein, side-chain cleavage, and 3β-hydroxysteroid dehydrogenase was significantly lower in SR-BI KD cells incubated with Fo. In conclusion, SR-BI protein deficiency, in part, might explain progesterone deficiency in some infertile women.

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