Separation of Lipid Transport Functions by Mutations in the Extracellular Domain of Scavenger Receptor Class B, Type I
2003; Elsevier BV; Volume: 278; Issue: 28 Linguagem: Inglês
10.1074/jbc.m302820200
ISSN1083-351X
AutoresMargery A. Connelly, Margarita de la Llera-Moya, Yinan Peng, Denise Drazul‐Schrader, George H. Rothblat, David L. Williams,
Tópico(s)Lipid Membrane Structure and Behavior
ResumoScavenger receptor class B, type I (SR-BI) shows a variety of effects on cellular cholesterol metabolism, including increased selective uptake of high density lipoprotein (HDL) cholesteryl ester, stimulation of free cholesterol (FC) efflux from cells to HDL and phospholipid vesicles, and changes in the distribution of plasma membrane FC as evidenced by increased susceptibility to exogenous cholesterol oxidase. Previous studies showed that these multiple effects require the extracellular domain of SR-BI, but not the transmembrane and cytoplasmic domains. To test whether 1) the extracellular domain of SR-BI mediates multiple activities by virtue of discrete functional subdomains, or 2) the multiple activities are, in fact, secondary to and driven by changes in cholesterol flux, the extracellular domain of SR-BI was subjected to insertional mutagenesis by strategically placing an epitope tag into nine sites. These experiments identified four classes of mutants with disruptions at different levels of function. Class 4 mutants showed a clear separation of function between HDL binding, HDL cholesteryl ester uptake, and HDL-dependent FC efflux on one hand and FC efflux to small unilamellar vesicles and an increased cholesterol oxidase-sensitive pool of membrane FC on the other. Selective disruption of the latter two functions provides evidence for multiple functional subdomains in the extracellular receptor domain. Furthermore, these findings uncover a difference in the SR-BI-mediated efflux pathways for FC transfer to HDL acceptors versus phospholipid vesicles. The loss of the cholesterol oxidase-sensitive FC pool and FC efflux to small unilamellar vesicle acceptors in Class 4 mutants suggests that these activities may be mechanistically related. Scavenger receptor class B, type I (SR-BI) shows a variety of effects on cellular cholesterol metabolism, including increased selective uptake of high density lipoprotein (HDL) cholesteryl ester, stimulation of free cholesterol (FC) efflux from cells to HDL and phospholipid vesicles, and changes in the distribution of plasma membrane FC as evidenced by increased susceptibility to exogenous cholesterol oxidase. Previous studies showed that these multiple effects require the extracellular domain of SR-BI, but not the transmembrane and cytoplasmic domains. To test whether 1) the extracellular domain of SR-BI mediates multiple activities by virtue of discrete functional subdomains, or 2) the multiple activities are, in fact, secondary to and driven by changes in cholesterol flux, the extracellular domain of SR-BI was subjected to insertional mutagenesis by strategically placing an epitope tag into nine sites. These experiments identified four classes of mutants with disruptions at different levels of function. Class 4 mutants showed a clear separation of function between HDL binding, HDL cholesteryl ester uptake, and HDL-dependent FC efflux on one hand and FC efflux to small unilamellar vesicles and an increased cholesterol oxidase-sensitive pool of membrane FC on the other. Selective disruption of the latter two functions provides evidence for multiple functional subdomains in the extracellular receptor domain. Furthermore, these findings uncover a difference in the SR-BI-mediated efflux pathways for FC transfer to HDL acceptors versus phospholipid vesicles. The loss of the cholesterol oxidase-sensitive FC pool and FC efflux to small unilamellar vesicle acceptors in Class 4 mutants suggests that these activities may be mechanistically related. Scavenger receptor class B, type I (SR-BI) 1The abbreviations used are: SR-BI, scavenger receptor class B, type I; HDL, high density lipoprotein; CE, cholesteryl ester; FC, free cholesterol; SUV, small unilamellar vesicle(s); PBS, phosphate-buffered saline; BSA, bovine serum albumin; PIPES, piperazine-N,N′-bis(2-ethanesulfonic acid); DLT, dilactitol tyramine; COE, cholesteryl oleyl ether. is an ∼82-kDa cell-surface glycoprotein that was first identified by its sequence homology to CD36 (1Calvo D. Vega M.A. J. Biol. Chem. 1993; 268: 18929-18935Abstract Full Text PDF PubMed Google Scholar, 2Acton S.L. Scherer P.E. Lodish H.F. Krieger M. J. Biol. Chem. 1994; 269: 21003-21009Abstract Full Text PDF PubMed Google Scholar) and later characterized as one of the first physiologically relevant receptors for high density lipoprotein (HDL) particles (Ref. 3Acton S. Rigotti A. Landschulz K.T. Xu S. Hobbs H.H. Krieger M. Science. 1996; 271: 518-520Crossref PubMed Scopus (2011) Google Scholar; for review, see Refs. 4Krieger M. Annu. Rev. Biochem. 1999; 68: 523-558Crossref PubMed Scopus (460) Google Scholar, 5Williams D.L. Connelly M.A. Temel R.E. Swarnakar S. Phillips M.C. de la Llera-Moya M. Rothblat G.H. Curr. Opin. Lipidol. 1999; 10: 329-339Crossref PubMed Scopus (173) Google Scholar, 6Silver D.L. Tall A.R. Curr. Opin. Lipidol. 2001; 12: 497-504Crossref PubMed Scopus (75) Google Scholar). Early analyses of SR-BI knockout mice revealed altered plasma HDL metabolism and reduced adrenal gland cholesteryl ester (CE) accumulation (7Rigotti A. Trigatti B.L. Penman M. Rayburn H. Herz J. Krieger M. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 12610-12615Crossref PubMed Scopus (761) Google Scholar, 8Rigotti A. Trigatti B. Babitt J. Penman M. Xu S. Krieger M. Curr. Opin. Lipidol. 1997; 8: 181-188Crossref PubMed Scopus (177) Google Scholar). 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SR-BI mediates its effects on HDL CE metabolism by facilitating the transport of lipids to cells in a process termed selective uptake (3Acton S. Rigotti A. Landschulz K.T. Xu S. Hobbs H.H. Krieger M. Science. 1996; 271: 518-520Crossref PubMed Scopus (2011) Google Scholar, 4Krieger M. Annu. Rev. Biochem. 1999; 68: 523-558Crossref PubMed Scopus (460) Google Scholar, 5Williams D.L. Connelly M.A. Temel R.E. Swarnakar S. Phillips M.C. de la Llera-Moya M. Rothblat G.H. Curr. Opin. Lipidol. 1999; 10: 329-339Crossref PubMed Scopus (173) Google Scholar, 18Glass C. Pittman R.C. Civen M. Steinberg D. J. Biol. Chem. 1985; 260: 744-750Abstract Full Text PDF PubMed Google Scholar, 19Reaven E. Chen Y.-D.I. Spicher M. Azhar S. J. Clin. Invest. 1984; 74: 1384-1397Crossref PubMed Scopus (94) Google Scholar, 20Gwynne J. Hess B. J. Biol. Chem. 1980; 255: 10875-10883Abstract Full Text PDF PubMed Google Scholar, 21Glass C. Pittman R.C. Weinstein D.B. Steinberg D. Proc. Natl. Acad. Sci. U. S. 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Cell Biol. 1985; 1: 1-39Crossref PubMed Scopus (1119) Google Scholar), whereas HDL CE delivered by SR-BI is hydrolyzed extralysosomally (26Sparrow C.P. Pittman R.C. Biochim. Biophys. Acta. 1990; 1043: 203-210Crossref PubMed Scopus (85) Google Scholar) by a neutral CE hydrolase (27DeLamatre J.G. Carter R.M. Hornick C.A. J. Cell. Physiol. 1993; 157: 164-168Crossref PubMed Scopus (29) Google Scholar, 28Shimada A. Tamai T. Oida K. Takahashi S. Suzuki J. Nakai T. Miyabo S. Biochim. Biophys. Acta. 1994; 1215: 126-132Crossref PubMed Scopus (26) Google Scholar). In fact, SR-BI has been shown to deliver HDL CE into a metabolically active membrane pool, where it is efficiently hydrolyzed by cell type-specific neutral CE hydrolases (29Connelly M.A. Kellner-Weiber G. Rothblat G.H. Williams D.L. J. Lipid Res. 2003; 44: 331-341Abstract Full Text Full Text PDF PubMed Scopus (48) Google Scholar). In addition to the uptake and metabolism of HDL CE, SR-BI stimulates the bidirectional flux of free cholesterol (FC) between cultured cells and lipoproteins (30Ji Y. Jian B. Wang N. Sun Y. de la Llera-Moya M. Phillips M.C. Rothblat G.H. Swaney J.B. Tall A.R. J. Biol. Chem. 1997; 272: 20982-20985Abstract Full Text Full Text PDF PubMed Scopus (636) Google Scholar, 31de la Llera-Moya M. Rothblat G.H. Connelly M.A. Kellner-Weibel G. Sakar S.W. Phillips M.C. Williams D.L. J. Lipid Res. 1999; 40: 575-580Abstract Full Text Full Text PDF PubMed Google Scholar, 32Jian B. de la Llera-Moya M. Ji Y. Wang N. Phillips M.C. Swaney J.B. Tall A.R. Rothblat G.H. J. Biol. Chem. 1998; 273: 5599-5606Abstract Full Text Full Text PDF PubMed Scopus (263) Google Scholar, 33Rothblat G.H. de la Llera-Moya M. Atger V. Kellner-Weiber G. Williams D.L. Phillips M.C. J. Lipid Res. 1999; 40: 781-796Abstract Full Text Full Text PDF PubMed Google Scholar, 34Stangl H. Hyatt M. Hobbs H.H. J. Biol. Chem. 1999; 274: 32692-32698Abstract Full Text Full Text PDF PubMed Scopus (124) Google Scholar), an activity that may be responsible for net cholesterol efflux from peripheral cells as well as the rapid hepatic clearance of FC from plasma HDL and its resultant secretion into bile (9Kozarsky K.F. Donahee M.H. Rigotti A. Iqbal S.N. Edelman E.R. Krieger M. Nature. 1997; 387: 414-417Crossref PubMed Scopus (631) Google Scholar, 35Ji Y. Wang N. Ramakrishnan R. Sehayek E. Huszar D. Breslow J.L. Tall A.R. J. Biol. Chem. 1999; 274: 33398-33402Abstract Full Text Full Text PDF PubMed Scopus (239) Google Scholar). SR-BI also increases cellular cholesterol mass and alters cholesterol distribution in plasma membrane domains as judged by the enhanced sensitivity of membrane cholesterol to extracellular cholesterol oxidase (31de la Llera-Moya M. Rothblat G.H. Connelly M.A. Kellner-Weibel G. Sakar S.W. Phillips M.C. Williams D.L. J. Lipid Res. 1999; 40: 575-580Abstract Full Text Full Text PDF PubMed Google Scholar, 36Kellner-Weibel G. de la Llera-Moya M. Connelly M.A. Stoudt G. Christian A.E. Haynes M.P. Williams D.L. Rothblat G.H. Biochemistry. 2000; 39: 221-229Crossref PubMed Scopus (133) Google Scholar). Moreover, the delivery of HDL FC by SR-BI results in efficient delivery of FC for esterification (36Kellner-Weibel G. de la Llera-Moya M. Connelly M.A. Stoudt G. Christian A.E. Haynes M.P. Williams D.L. Rothblat G.H. Biochemistry. 2000; 39: 221-229Crossref PubMed Scopus (133) Google Scholar, 37Connelly M.A. de la Llera-Moya M. Monzo P. Yancey P. Drazul D. Stoudt G. Fournier N. Klein S.M. Rothblat G.H. Williams D.L. Biochemistry. 2001; 40: 5249-5259Crossref PubMed Scopus (72) Google Scholar). Together, these data support the idea that, similar to HDL CE, SR-BI delivers HDL FC into a metabolically active membrane pool. Whether this membrane pool reflects the localization of SR-BI in a physically distinct membrane domain or its interaction with other membrane proteins is not known. Previous studies have shown that several key lipid transport functions of SR-BI, including the ability to alter membrane cholesterol domains, are dependent on its extracellular region (37Connelly M.A. de la Llera-Moya M. Monzo P. Yancey P. Drazul D. Stoudt G. Fournier N. Klein S.M. Rothblat G.H. Williams D.L. Biochemistry. 2001; 40: 5249-5259Crossref PubMed Scopus (72) Google Scholar, 38Gu X. Trigatti B. Xu S. Acton S. Babitt J. Krieger M. J. Biol. Chem. 1998; 273: 26338-26348Abstract Full Text Full Text PDF PubMed Scopus (200) Google Scholar, 39Connelly M.A. Klein S.M. Azhar S. Abumrad N.A. Williams D.L. J. Biol. Chem. 1999; 274: 41-47Abstract Full Text Full Text PDF PubMed Scopus (193) Google Scholar). However, to date, it is unclear whether 1) the extracellular domain of SR-BI mediates multiple distinct activities by virtue of discrete functional domains or 2) the distinct functions of SR-BI are, in fact, secondary to, and driven by, changes in cellular cholesterol content and distribution. To address this question, we mutagenized the extracellular domain of murine SR-BI by strategically placing an epitope tag from the adenovirus E4/5 protein into nine sites in the SR-BI extracellular domain (designated A-II through A-X in Fig. 1 and “Experimental Procedures”). We expressed the mutant receptors in COS-7 cells, checked for cell-surface expression of the mutant receptors, and assessed the ability of these epitope-tagged mutants to mediate multiple aspects of cellular lipid metabolism. These experiments identified four classes of SR-BI mutants that revealed that 1) all SR-BI activities do not derive from the ability of SR-BI to load cholesterol into the plasma membrane, and 2) there is a separation of function between SR-BI-mediated HDL CE uptake and HDL FC efflux on one hand and FC efflux to small unilamellar vesicle (SUV) acceptors and an increased cholesterol oxidase-sensitive pool of membrane FC on the other. These data provide clear evidence for a difference in the pathways for SR-BI-mediated FC efflux to HDL and to phospholipid vesicles. Materials—The following antibodies were used: anti-M45 epitope hybridoma medium (a generous gift from Dr. Patrick Hearing, State University of New York, Stony Brook; 1:100 for immunofluorescence, 1:250 for flow cytometry, and 1:500 for immunoblotting) (40Obert S. O'Connor R.J. Schmid S. Hearing P. Mol. Cell. Biol. 1994; 14: 1333-1346Crossref PubMed Scopus (118) Google Scholar), anti-His6 monoclonal IgG (Roche Applied Science; 1:500 for immunoblotting), anti-SR-BI extracellular domain polyclonal antibody 356 (directed against residues 174–356; 1:250 for flow cytometry) (41Temel R.E. Trigatti B. DeMattos R.B. Azhar S. Krieger M. Williams D.L. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 13600-13605Crossref PubMed Scopus (210) Google Scholar), anti-SR-BI C-terminal domain polyclonal antibody (Novus Biologicals, Inc.; 1:5000 for immunoblotting), anti-SR-BI extracellular domain polyclonal antibody (Novus Biologicals, Inc.; 1:500 for immunofluorescence), peroxidase-conjugated goat anti-mouse or anti-rabbit secondary IgG (Jackson ImmunoResearch Laboratories, Inc.; 1:10,000 for immunoblotting), fluorescein (Sigma)- or phycoerythrin (Molecular Probes, Inc.)-conjugated anti-rabbit antibody (1:100 for flow cytometry), and Alexa 488-conjugated goat anti-mouse or anti-rabbit secondary IgG (Molecular Probes, Inc.; 1:1000 for immunofluorescence). Plasmids and Sequencing—PCR amplifications were performed using a PerkinElmer Life Sciences DNA Thermal Cycler 9700. Oligonucleotides were purchased from Integrated DNA Technologies. The “seamless cloning” technique from Stratagene was modified and employed to clone monoclonal antibody epitopes into the extracellular domain of murine SR-BI. For construction of A-II, primers 5′-AGCCAGCTCTTCACTACCTCCTTTTGAGACAGAGACGATCCTGTGGGGCTATGACGATC-3′ and 5′-AGCTAGCTCTTCATAGGCGATCCCTACTCCGATCCTCACCAACTGTGCGGTTC-3′ were employed to amplify the entire pSG5(mSR-BI) plasmid. The resulting PCR product was digested with SapI (New England Biolabs Inc.) and recircularized. This resulted in the insertion of a 14-amino acid M45 monoclonal antibody epitope (DRSRDRLPPFETET) (40Obert S. O'Connor R.J. Schmid S. Hearing P. Mol. Cell. Biol. 1994; 14: 1333-1346Crossref PubMed Scopus (118) Google Scholar) into SR-BI, C-terminal to amino acid 179. For construction of A-III, primers 5′-AGCCAGCTCTTCACTACCTCCTTTTGAGACAGAGACGAAGCTGACCTACAACGAATC-3′ and 5′-AGCTAGCTCTTCATAGGCGATCCCTACTCCGATCCATGGACCTGCATGCCTC-3′ were employed to insert the M45 epitope C-terminal to amino acid 283. For construction of A-IV, primers 5′-AGCCAGCTCTTCACTACCTCCTTTTGAGACAGAGACGCAGCTGAGCCTCTACATCAAATCTGTC-3′ and 5′-AGCTAGCTCTTCATAGGCGATCCCTACTCCGATCCATCTTCACAGAACAGTTCATGGGG-3′ were employed to insert the M45 epitope C-terminal to amino acid 388. For construction of A-V, primers 5′-AGCCAGCTCTTCACTACCTCCTTTTGAGACAGAGACGCTCTCCCACCCCCACTTTTAC-3′ and 5′-AGCTAGCTCTTCATAGGCGATCCCTACTCCGATCAAACAGAGGCGCACCAAAC-3′ were employed to insert the M45 epitope C-terminal to amino acid 341. For construction of A-VI, primers 5′-AGCCAGCTCTTCACTACCTCCTTTTGAGACAGAGACGAAGCCCCTGAGCACGTTCTAC-3′ and 5′-AGCCAGCTCTTCATAGCCTGTCCCTACTCCGATCGCCACCCATTGCTCCGCTCTG-3′ were employed to insert the M45 epitope C-terminal to amino acid 424. For construction of A-VII, primers 5′-AGCCAGCTCTTCACTACCTCCTTTTGAGACAGAGACGTCAAGGGTGTTTGAAGGCATTC-3′ and 5′-AGCCAGCTCTTCATAGGCGATCCCTACTCCGATCTTCGTTGTAGGTCAGCTTCATGG-3′ were employed to insert the M45 epitope C-terminal to amino acid 289. For construction of A-VIII, primers 5′-AGCCAGCTCTTCACTACCTCCTTTTGAGACAGAGACGACGTACCTCCCAGACATGCTTC-3′ and 5′-AGCGAGCTCTTCATAGGCTATCCCTACTCCGATCATTGAGAAAATGCACGAAGGG-3′ were employed to insert the M45 epitope C-terminal to amino acid 192. For construction of A-IX, primers 5′-AGCCAGCTCTTCACTACCTCCTTTTGAGACAGAGACGAACGGGCAGAAGCCAGTAGTC-3′ and 5′-AGCCAGCTCTTCATAGTCTATCCCTACTCCGATCGAGGACCTCGTTTGGGTTGAC-3′ were employed to insert the M45 epitope C-terminal to amino acid 79. For construction of A-X, primers 5′-AGCCAGCTCTTCACTACCTCCTTTTGAGACAGAGACGAGCAGCCTGTCCTTCGGG-3′ and 5′-AGCCAGCTCTTCATAGGCGATCCCTACTCCGATCCGGGTCTATGCGGACATTC-3′ were employed to insert the M45 epitope C-terminal to amino acid 48. For construction of H-VI, primers 5′-AGCTAGCTCTTCACATCATCACACGTTCTACACGCAGCTGGTG-3′ and 5′-AGCTAGCTCTTCTATGATGGTGATGCATTGCTCCGCTCTGTTCG-3′ were employed to replace amino acids 423–428 of SR-BI with six histidines. This created an anti-histidine monoclonal antibody epitope (HHHHHH). All plasmids were prepared using endotoxin-free QIAGEN maxi-prep kits and sequenced throughout the SR-BI coding region to confirm the correct epitope insertion and to ensure that no point mutations had been generated during the amplification process. DNA sequencing was performed by the automated sequencing facility at the State University of New York (Stony Brook). Reactions were prepared using a dye termination cycle sequencing kit and analyzed on an Applied Biosystems Model 373 DNA Sequencer with an Excel upgrade as recommended by the manufacturer (PE Applied Biosystems). Transient Transfection of COS-7 Cells—COS-7 cells were maintained in Dulbecco's modified Eagle's medium (Invitrogen), 10% calf serum (Atlanta Biologicals, Inc.), 2 mm l-glutamine, 50 units/ml penicillin, 50 μg/ml streptomycin, and 1 mm sodium pyruvate and transfected as described previously (39Connelly M.A. Klein S.M. Azhar S. Abumrad N.A. Williams D.L. J. Biol. Chem. 1999; 274: 41-47Abstract Full Text Full Text PDF PubMed Scopus (193) Google Scholar). The following day, two 10-cm dishes of transfected cells were trypsinized and resuspended in a total volume of 12 ml with fresh medium, and 0.5 or 1 ml was dispensed to each 22-mm (12-well plate) or 35-mm (6-well plate) well, respectively (one 10-cm dish is equivalent to one 12-well or one 6-well plate). The cells were assayed 48 h post-transfection unless otherwise indicated. Immunoblot Analysis—Transiently transfected cells expressing SR-BI (in 35-mm wells) were washed twice with phosphate-buffered saline (PBS; Invitrogen), pH 7.4, and lysed with 300 μl of Nonidet P-40 cell lysis buffer (41Temel R.E. Trigatti B. DeMattos R.B. Azhar S. Krieger M. Williams D.L. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 13600-13605Crossref PubMed Scopus (210) Google Scholar, 42Rigotti A. Edelman E.R. Seifert P. Iqbal S.N. DeMattos R.B. Temel R.E. Krieger M. Williams D.L. J. Biol. Chem. 1996; 271: 33545-33549Abstract Full Text Full Text PDF PubMed Scopus (205) Google Scholar) containing 1 μg/ml pepstatin, 0.2 mm phenylmethylsulfonyl fluoride, 1 μg/ml leupeptin, and 10 μg/ml aprotinin. Protein concentrations were determined by the method of Lowry et al. (43Lowry O.H. Rosebrough N.J. Farr A.L. Randall R.J. J. Biol. Chem. 1951; 193: 265-275Abstract Full Text PDF PubMed Google Scholar). Immunoblots with antibodies directed to SR-BI confirmed mutant and wild-type receptor expression. Equal amounts of total cell proteins were separated on precast 10% SDS-polyacrylamide gels (Bio-Rad), blotted onto nitrocellulose membranes (Bio-Rad), and detected using antibody directed against the C-terminal domain of SR-BI or the respective monoclonal antibody epitope, horseradish peroxidase-conjugated anti-rabbit secondary antibody, and SuperSignal West Pico reagent (Pierce). Blots were quantified using a Bio-Rad Model GS-700 imaging densitometer and MultiAnalyst software. Cell-surface Receptor Expression Levels by Flow Cytometry—Transiently transfected COS-7 cells (in 35-mm wells) were washed with 2 ml of cold PBS. Cells were removed from plates by the addition of 1 ml of PBS and 0.5 mm EDTA and incubation for 5–7 min at room temperature. Cells were placed in a microcentrifuge tube, centrifuged at 200 × g for 2–3 min, and resuspended in 100 μl of PBS and 1% bovine serum albumin (BSA). Anti-SR-BI primary antibody 356 at a concentration of 0.48 mg/ml IgG or anti-M45 hybridoma medium at a 1:250 dilution was added to the cells and incubated for 1 h at 4 °C. The cells were centrifuged at 200 × g for 2–3 min, and the supernatant was aspirated. Cells were washed twice with 0.5 ml of PBS and 1% BSA before incubation with secondary antibody (3 μl of fluorescein- or phycoerythrin-conjugated anti-rabbit antibody) in 300 μl of PBS and 1% BSA for 30 min at 4 °C. Cells were washed three times with 0.5 ml of PBS and 1% BSA and fixed in 0.5 ml of 1% formaldehyde in PBS and 1% BSA for 15 min at 4 °C with gently shaking. Following incubation with fixative, the cells were centrifuged at 200 × g for 2–3 min and resuspended in 0.5 ml of PBS and 1% BSA. Fluorescence intensities were measured using a BD Biosciences FACS-Advantage cell sorter or a FACScan flow cytometer. Immunofluorescence—Transiently transfected COS-7 cells were replated 24 h post-transfection onto a 12-well plate containing glass microscope slide coverslips. After 24 h, the medium was removed, and the cells were washed at room temperature with PBS. The cells were fixed for1hin4% (w/v) paraformaldehyde in 77 mm PIPES, pH 7.5 (44Berrios M. Conlon K.A. Colflesh D.E. Methods Enzymol. 1999; 307: 55-79Crossref PubMed Scopus (14) Google Scholar); washed with PBS; and blocked for 1 h with 3% BSA and 10 mm glycine in PBS. Antibody against the extracellular domain of SR-BI was diluted in 3% BSA and applied to cells for 1 h at room temperature. Cells were washed as described above and incubated with Alexa 488-conjugated secondary antibodies for 30 min at room temperature. Cells were then washed with PBS and mounted using ProLong antifade mounting medium (Molecular Probes, Inc.). Cells were examined using a Leica DMIRE2 confocal microscope, and images were collected using Leica confocal software. Preparation of 125I-Dilactitol Tyramine (DLT)- and [3H]Cholesteryl Oleyl Ether (COE)-labeled HDL and 125I-HDL—Human HDL3 (1.125 g/ml < ρ < 1.210 g/ml), herein referred to as HDL, was isolated by sequential ultracentrifugation (45Havel R.J. Eder H.A. Bragdon J.H. J. Clin. Invest. 1955; 34: 1345-1353Crossref PubMed Scopus (6498) Google Scholar). The HDL was labeled with [3H]COE (Amersham Biosciences) using recombinant CE transfer protein (Cardiovascular Targets, Inc.) as described (46Francone O.L. Haghpassand M. Bennett J.A. Royer L. McNeish J. J. Lipid Res. 1997; 38: 813-822Abstract Full Text PDF PubMed Google Scholar) with the following modifications. HDL and CE transfer protein were incubated with [3H]COE (dried down on the glass vial) for 5 h at 37 °C. Labeled particles were reisolated by gel exclusion chromatography on a 25-ml Superose 6 column (Amersham Biosciences). The HDL was then labeled with 125I-DLT as described previously (39Connelly M.A. Klein S.M. Azhar S. Abumrad N.A. Williams D.L. J. Biol. Chem. 1999; 274: 41-47Abstract Full Text Full Text PDF PubMed Scopus (193) Google Scholar). Particles were dialyzed against four changes of 150 mm NaCl, 10 mm potassium phosphate buffer, pH 7.4, and 1 mm EDTA and stored at 4 °C under argon. The average specific activity of the 125I-DLT- and [3H]COE-labeled HDL was 650 dpm/ng of protein for 125I and 37 dpm/ng of protein for 3H. For some experiments, HDL was labeled using the iodine monochloride method (47Goldstein J. Basu S.K. Brown M. Methods Enzymol. 1983; 98: 241-260Crossref PubMed Scopus (1287) Google Scholar), and the average specific activity of the 125I-HDL was ∼500 dpm/ng of protein. HDL Cell Association, Selective COE Uptake, and Apolipoprotein Degradation—Transiently transfected COS-7 cells (in 35-mm wells) were washed once with serum-free Dulbecco's modified Eagle's medium and 0.5% BSA. 125I-DLT- and [3H]COE-labeled HDL particles were added at a concentration of 10 μg/ml protein (unless otherwise indicated) in serum-free Dulbecco's modified Eagle's medium and 0.5% BSA. After incubation for 1.5 h at 37 °C, the medium was removed, and the cells were washed three times with PBS and 0.1% BSA, pH 7.4, and one time with PBS, pH 7.4. The cells were lysed with 1.1 ml of 0.1 n NaOH, and the lysate was processed to determine trichloroacetic acidsoluble and -insoluble 125I radioactivity and organic solvent-extractable 3H radioactivity. The values for cell-associated HDL apolipoprotein, total cell-associated HDL COE, and the selective uptake of HDL COE were obtained as described previously (39Connelly M.A. Klein S.M. Azhar S. Abumrad N.A. Williams D.L. J. Biol. Chem. 1999; 274: 41-47Abstract Full Text Full Text PDF PubMed Scopus (193) Google Scholar). Cholesterol Efflux Assay—Transiently transfected COS-7 cells were replated onto 11-mm wells in growth medium. Cells were labeled for 24 h with 5 μCi/ml [3H]cholesterol (PerkinElmer Life Sciences) in Dulbecco's modified Eagle's medium containing 10% calf serum immediately after reseeding. Cells were washed, and [3H]cholesterol efflux was measured at 2 h in triplicate using different concentrations of HDL acceptor or palmitoyloleoylphosphatidylcholine-containing SUV as described previously (31de la Llera-Moya M. Rothblat G.H. Connelly M.A. Kellner-Weibel G. Sakar S.W. Phillips M.C. Williams D.L. J. Lipid Res. 1999; 40: 575-580Abstract Full Text Full Text PDF PubMed Google Scholar). The release of radioactive cholesterol was measured by scintillation counting of filtered aliquots of acceptor-containing medium and expressed as the fraction of the total 2-propanol-soluble label in the cells plus the label that was released into the medium. Fractional efflux values were corrected for the small amount of radioactivity released in the absence of acceptor. To normalize the data for FC efflux to the amount of cell-surface receptor expressed in the transient transfections, modified HDL cell association assays were performed in parallel with the FC efflux studies. COS-7 cells (in 22-mm wells) were washed once with serum-free minimal essential medium/HEPES and 1% BSA, and monochloride-labeled 125I-HDL was added at 10 or 25 μg of protein/ml of minimal essential medium/HEPES. After incu
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