PMID- 22097902
OWN - NLM
STAT- MEDLINE
DCOM- 20120213
LR  - 20211021
IS  - 1520-4995 (Electronic)
IS  - 0006-2960 (Print)
IS  - 0006-2960 (Linking)
VI  - 50
IP  - 50
DP  - 2011 Dec 20
TI  - Differential roles of cysteine residues in the cellular trafficking, 
      dimerization, and function of the high-density lipoprotein receptor, SR-BI.
PG  - 10860-75
LID - 10.1021/bi201264y [doi]
AB  - The scavenger receptor, class B, type I (SR-BI) binds high-density lipoprotein 
      (HDL) and mediates selective delivery of cholesteryl esters (CEs) to the liver 
      and steroidogenic cells of the adrenal glands and gonads. Although it is clear 
      that the large extracellular domain (ECD) of SR-BI binds HDL, the role of ECD in 
      the selective HDL-CE transport remains poorly understood. In this study, we used 
      a combination of mutational and chemical approaches to systematically evaluate 
      the contribution of cysteine residues, especially six cysteine residues of ECD, 
      in SR-BI-mediated selective HDL-CE uptake, intracellular trafficking, and SR-BI 
      dimerization. Pretreatment of SR-BI-overexpressing COS-7 cells with a disulfide 
      (S-S) bond reducing agent, beta-mercaptoethanol (100 mM) or dithiothreitol (DTT) (10 
      mM), modestly but significantly impaired SR-BI-mediated selective HDL-CE uptake. 
      Treatment of SR-BI-overexpressing COS-7 cells with the optimal doses of membrane 
      permeant alkyl methanethiosulfonate (MTS) reagents, positively charged MTSEA or 
      neutral MMTS, that specifically react with the free sulfhydryl group of cysteine 
      reduced the rate of SR-BI-mediated selective HDL-CE uptake, indicating that 
      certain intracellular free cysteine residues may also be critically involved in 
      the selective cholesterol transport process. In contrast, use of membrane 
      impermeant MTS reagent, positively charged MTSET and negatively charged MTSES, 
      showed no such effect. Next, the importance of eight cysteine residues in SR-BI 
      expression, cell surface expression, dimer formation, and selective HDL-derived 
      CE transport was evaluated. These cysteine residues were replaced either singly 
      or in pairs with serine, and the mutant SR-BIs were expressed in either COS-7 or 
      CHO cells. Four mutations, C280S, C321S, C323S, and C334S, of the ECD, either 
      singly or in various pair combinations, resulted in significant decreases in 
      SR-BI (HDL) binding activity, selective CE uptake, and trafficking to the cell 
      surface. Surprisingly, we found that mutation of the two remaining cysteine 
      residues, C251 and C384 of the ECD, had no effect on either SR-BI expression or 
      function. Other cysteine mutations and substitutions were also without effect. 
      Western blot data indicated that single and double mutations at C280, C321, C323, 
      and C334 residues strongly favor dimer formation. However, they are rendered 
      nonfunctional presumably because of mutation-induced formation of aberrant 
      disulfide linkages resulting in inhibition of optimal HDL binding and, thus, 
      selective HDL-CE uptake. These results provide novel insights into the functional 
      role of four cysteine residues, C280, C321, C323, and C334, of the SR-BI ECD in 
      SR-BI expression and trafficking to the cell surface, its dimerization, and 
      associated selective CE transport function.
FAU - Hu, Jie
AU  - Hu J
AD  - Geriatric Research, Education and Clinical Center, VA Palo Alto Health Care 
      System, Palo Alto, California 94304, United States.
FAU - Zhang, Zhonghua
AU  - Zhang Z
FAU - Shen, Wen-Jun
AU  - Shen WJ
FAU - Nomoto, Ann
AU  - Nomoto A
FAU - Azhar, Salman
AU  - Azhar S
LA  - eng
GR  - R01 HL033881/HL/NHLBI NIH HHS/United States
GR  - R01 HL033881-26/HL/NHLBI NIH HHS/United States
GR  - HL033881/HL/NHLBI NIH HHS/United States
PT  - Journal Article
PT  - Research Support, N.I.H., Extramural
PT  - Research Support, U.S. Gov't, Non-P.H.S.
DEP - 20111129
PL  - United States
TA  - Biochemistry
JT  - Biochemistry
JID - 0370623
RN  - 0 (Cholesterol Esters)
RN  - 0 (HDL cholesteryl ester)
RN  - 0 (Lipoproteins, HDL)
RN  - 0 (Mutant Proteins)
RN  - 0 (Receptors, Lipoprotein)
RN  - 0 (Recombinant Proteins)
RN  - 0 (Reducing Agents)
RN  - 0 (SCARB1 protein, human)
RN  - 0 (Scavenger Receptors, Class B)
RN  - 0 (high density lipoprotein receptors)
RN  - K848JZ4886 (Cysteine)
SB  - IM
MH  - Alkylation
MH  - Amino Acid Substitution
MH  - Animals
MH  - CHO Cells
MH  - COS Cells
MH  - Chlorocebus aethiops
MH  - Cholesterol Esters/metabolism
MH  - Cricetinae
MH  - Cricetulus
MH  - Cysteine/*chemistry
MH  - Dimerization
MH  - Down-Regulation
MH  - Lipoproteins, HDL/chemistry/genetics/*metabolism
MH  - Mutagenesis, Site-Directed
MH  - Mutant Proteins/chemistry/metabolism
MH  - Oxidation-Reduction
MH  - Protein Structure, Tertiary
MH  - Protein Transport
MH  - Receptors, Lipoprotein/chemistry/genetics/metabolism
MH  - Recombinant Proteins/chemistry/metabolism
MH  - Reducing Agents/pharmacology
MH  - Scavenger Receptors, Class B/*chemistry/genetics/*metabolism
PMC - PMC3262050
MID - NIHMS341396
EDAT- 2011/11/22 06:00
MHDA- 2012/02/14 06:00
PMCR- 2012/12/20
CRDT- 2011/11/22 06:00
PHST- 2011/11/22 06:00 [entrez]
PHST- 2011/11/22 06:00 [pubmed]
PHST- 2012/02/14 06:00 [medline]
PHST- 2012/12/20 00:00 [pmc-release]
AID - 10.1021/bi201264y [doi]
PST - ppublish
SO  - Biochemistry. 2011 Dec 20;50(50):10860-75. doi: 10.1021/bi201264y. Epub 2011 Nov 
      29.