Effects of amino acid substitutions at glycine 420 on SR-BI cholesterol transport function
2007; Elsevier BV; Volume: 48; Issue: 6 Linguagem: Inglês
10.1194/jlr.m700086-jlr200
ISSN1539-7262
AutoresSaj Parathath, Yolanda F. Darlington, Margarita de la Llera Moya, Denise Drazul‐Schrader, David L. Williams, Michael C. Phillips, George H. Rothblat, Margery A. Connelly,
Tópico(s)Metabolomics and Mass Spectrometry Studies
ResumoScavenger receptor class B type I (SR-BI) facilitates the uptake of HDL cholesteryl esters (CEs) in a two-step process involving binding of HDL to its extracellular domain and transfer of HDL core CEs to a metabolically active membrane pool, where they are subsequently hydrolyzed by a neutral CE hydrolase. Recently, we characterized a mutant, G420H, which replaced glycine 420 in the extracellular domain of SR-BI with a histidine residue and had a profound effect on SR-BI function. The G420H mutant receptor exhibited a reduced ability to mediate selective HDL CE uptake and was unable to deliver HDL CE for hydrolysis, despite the fact that it retained the ability to bind HDL. This did not hold true if glycine 420 was replaced with an alanine residue; G420A maintained wild-type HDL binding and cholesterol transport activity. To further understand the role that glycine 420 plays in SR-BI function and why there was a disparity between replacing glycine 420 with a histidine versus an alanine, we generated a battery of point mutants by substituting glycine 420 with amino acids possessing side chains that were charged, hydrophobic, polar, or bulky and tested the resulting mutants for their ability to support HDL binding, HDL cholesterol transport, and delivery for hydrolysis. The results indicated that substitution with a negatively charged residue or a proline impaired cell surface expression of SR-BI or its interaction with HDL, respectively. Furthermore, substitution of glycine 420 with a positively charged residue reduced HDL CE uptake as well as its subsequent hydrolysis. Scavenger receptor class B type I (SR-BI) facilitates the uptake of HDL cholesteryl esters (CEs) in a two-step process involving binding of HDL to its extracellular domain and transfer of HDL core CEs to a metabolically active membrane pool, where they are subsequently hydrolyzed by a neutral CE hydrolase. Recently, we characterized a mutant, G420H, which replaced glycine 420 in the extracellular domain of SR-BI with a histidine residue and had a profound effect on SR-BI function. The G420H mutant receptor exhibited a reduced ability to mediate selective HDL CE uptake and was unable to deliver HDL CE for hydrolysis, despite the fact that it retained the ability to bind HDL. This did not hold true if glycine 420 was replaced with an alanine residue; G420A maintained wild-type HDL binding and cholesterol transport activity. To further understand the role that glycine 420 plays in SR-BI function and why there was a disparity between replacing glycine 420 with a histidine versus an alanine, we generated a battery of point mutants by substituting glycine 420 with amino acids possessing side chains that were charged, hydrophobic, polar, or bulky and tested the resulting mutants for their ability to support HDL binding, HDL cholesterol transport, and delivery for hydrolysis. The results indicated that substitution with a negatively charged residue or a proline impaired cell surface expression of SR-BI or its interaction with HDL, respectively. Furthermore, substitution of glycine 420 with a positively charged residue reduced HDL CE uptake as well as its subsequent hydrolysis. apolipoprotein A-I blocks lipid transport-1 cholesteryl ester cholesteryl oleyl ether free cholesterol scavenger receptor class B type I Scavenger receptor class B type I (SR-BI) is a physiologically relevant HDL receptor that participates in many aspects of HDL cholesterol metabolism (for review, see Ref. 1.Rhainds D. Brissette L. The role of scavenger receptor class B type I (SR-BI) in lipid trafficking. Defining the rules for lipid traders.Int. J. Biochem. Cell Biol. 2004; 36: 39-77Crossref PubMed Scopus (131) Google Scholar, 2.Connelly M.A. Williams D.L. Scavenger receptor BI: a scavenger receptor with a mission to transport high density lipoprotein lipids.Curr. Opin. Lipidol. 2004; 15: 287-295Crossref PubMed Scopus (134) Google Scholar, 3.Connelly M.A. Williams D.L. SR-BI and HDL cholesteryl ester metabolism.Endocr. 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Expression and microvillar localization of scavenger receptor, class B, type I (a high density lipoprotein receptor) in luteinized and hormone-desensitized rat ovarian models.Endocrinology. 1998; 139: 2847-2856Crossref PubMed Scopus (0) Google Scholar, 13.Azhar S. Tsai L. Reaven E. Uptake and utilization of lipoprotein cholesteryl esters by rat granulosa cells.Biochim. Biophys. Acta. 1990; 1047: 148-160Crossref PubMed Scopus (61) Google Scholar, 14.Out R. Hoekstra M. Spijkers J.A. Kruijt J.K. Eck M.Van Bos I.S. Twisk J. Van Berkel T.J. Scavenger receptor class B type I is solely responsible for the selective uptake of cholesteryl esters from HDL by the liver and the adrenals in mice.J. Lipid Res. 2004; 45: 2088-2095Abstract Full Text Full Text PDF PubMed Scopus (108) Google Scholar, 15.Brodeur M.R. Luangrath V. Bourret G. Falstrault L. Brissette L. Physiological importance of SR-BI in the in vivo metabolism of human HDL and LDL in male and female mice.J. 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Additionally, SR-BI has been shown to facilitate the bidirectional flux of free cholesterol (FC) (19.Ji Y. Jian B. Wang N. Sun Y. de la Llera Moya M. Phillips M.C. Rothblat G.H. Swaney J.B. Tall A.R. Scavenger receptor BI promotes high density lipoprotein-mediated cellular cholesterol efflux.J. Biol. Chem. 1997; 272: 20982-20985Abstract Full Text Full Text PDF PubMed Scopus (633) Google Scholar, 20.Jian B. de la Llera-Moya M. Ji Y. Wang N. Phillips M.C. Swaney J.B. Tall A.R. Rothblat G.H. Scavenger receptor class B type I as a mediator of cellular cholesterol efflux to lipoproteins and phospholipid acceptors.J. Biol. Chem. 1998; 273: 5599-5606Abstract Full Text Full Text PDF PubMed Scopus (262) Google Scholar, 21.Rothblat G.H. de la Llera-Moya M. Atger V. Kellner-Weiber G. Williams D.L. Phillips M.C. Cell cholesterol efflux: integration of old and new observations provides new insights.J. Lipid Res. 1999; 40: 781-796Abstract Full Text Full Text PDF PubMed Google Scholar, 22.Zimetti F. Weibel G.K. Duong M. Rothblat G.H. Measurement of cholesterol bidirectional flux between cells and lipoproteins.J. Lipid Res. 2006; 47: 605-613Abstract Full Text Full Text PDF PubMed Scopus (67) Google Scholar). These observations suggest that SR-BI is important at both ends of the reverse cholesterol transport pathway: efflux of FC from cells within the vessel wall and selective uptake of HDL CE and FC into the liver. Although it is well established that SR-BI's role in the reverse cholesterol transport pathway is particularly important for cholesterol metabolism in rodents, it is not clear whether this is true in humans. However, several recent studies have reported genetic polymorphisms that implicate SR-BI as a significant player in human HDL cholesterol metabolism as well (23.Acton S. Osgood D. Donoghue M. Corella D. Pocovi M. Cenarro A. Mozas P. Keilty J. Squazzo S. Woolf E.A. et al.Association of polymorphisms at the SR-BI gene locus with plasma lipid levels and body mass index in a white population.Arterioscler. Thromb. Vasc. Biol. 1999; 19: 1734-1743Crossref PubMed Scopus (189) Google Scholar, 24.Hong S.H. Kim Y.R. Yoon Y.M. Min W.K. Chun S.I. Kim J.Q. Association between HaeIII polymorphism of scavenger receptor class B type I gene and plasma HDL-cholesterol concentration.Ann. Clin. Biochem. 2002; 39: 478-481Crossref PubMed Scopus (39) Google Scholar, 25.Osgood D. Corella D. Demissie S. Cupples L.A. Wilson P.W. Meigs J.B. Schaefer E.J. Coltell O. Ordovas J.M. Genetic variation at the scavenger receptor class B type I gene locus determines plasma lipoprotein concentrations and particle size and interacts with type 2 diabetes: the Framingham Study.J. Clin. Endocrinol. Metab. 2003; 88: 2869-2879Crossref PubMed Scopus (100) Google Scholar, 26.Hsu L.A. Ko Y.L. Wu S. Teng M.S. Peng T.Y. Chen C.F. Chen C.F. Lee Y.S. 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Postprandial lipemia is modified by the presence of the polymorphism present in the exon 1 variant at the SR-BI gene locus.J. Mol. Endocrinol. 2004; 32: 237-245Crossref PubMed Scopus (35) Google Scholar, 30.Rodriguez-Esparragon F. Rodriguez-Perez J.C. Hernandez-Trujillo Y. Macias-Reyes A. Medina A. Caballero A. Ferrario C.M. Allelic variants of the human scavenger receptor class B type 1 and paraoxonase 1 on coronary heart disease: genotype-phenotype correlations.Arterioscler. Thromb. Vasc. Biol. 2005; 25: 854-860Crossref PubMed Scopus (58) Google Scholar). SR-BI-mediated HDL CE selective uptake is a two-step process involving 1) lipoprotein binding to the extracellular domain of SR-BI and 2) lipid transfer from the HDL particle to the cell (31.Connelly M.A. Williams D.L. SR-BI and cholesterol uptake into steroidogenic cells.Trends Endocrinol. Metab. 2003; 14: 467-472Abstract Full Text Full Text PDF PubMed Scopus (59) Google Scholar, 32.Rigotti A.M.H. The role of the high-density lipoprotein receptor SR-BI in the lipid metabolism of endocrine and other tissues.Endocr. Rev. 2003; 24: 357-387Crossref PubMed Scopus (348) Google Scholar, 33.Vinals M. Xu S. Vasile E. Krieger M. Identification of the N-linked glycosylation sites on the high density lipoprotein (HDL) receptor SR-BI and assessment of their effects on HDL binding and selective lipid uptake.J. Biol. Chem. 2003; 278: 5325-5332Abstract Full Text Full Text PDF PubMed Scopus (63) Google Scholar). Recent reports showed conclusively that it is not only the presence but also the proper orientation of apolipoprotein A-I (apoA-I) that is necessary for optimal SR-BI-mediated HDL CE selective uptake (34.Liu T. Krieger M. Kan H.Y. Zannis V.I. The effects of mutations in helices 4 and 6 of apoA-I on SR-BI-mediated cholesterol efflux suggest that formation of a productive complex between reconstituted HDL and SR-BI is required for efficient lipid transport.J. Biol. Chem. 2002; 277: 21578-21584Google Scholar, 35.Temel R.E. Walzem R.L. Banka C.L. Williams D.L. Apolipoprotein A-I is necessary for the in vivo formation of HDL competent for scavenger receptor BI-mediated cholesteryl ester selective uptake.J. Biol. Chem. 2002; 277: 26565-26572Abstract Full Text Full Text PDF PubMed Scopus (47) Google Scholar, 36.Temel R.E. Parks J.S. Williams D.L. Enhancement of SR-BI-mediated cholesteryl ester selective uptake from apoA-I−/− HDL by apoA-I requires HDL reorganization by LCAT.J. Biol. Chem. 2003; 278: 4792-4799Abstract Full Text Full Text PDF PubMed Scopus (19) Google Scholar, 37.Thuahnai S.T. Lund-Katz S. Dhanasekaran P. Llera-Moya M. Connelly M.A. Williams D.L. Rothblat G.H. Phillips M.C. Scavenger receptor class B type I-mediated cholesteryl ester-selective uptake and efflux of unesterified cholesterol. Influence of high density lipoprotein size and structure.J. Biol. Chem. 2004; 279: 12448-12455Abstract Full Text Full Text PDF PubMed Scopus (80) Google Scholar). In addition, BLT-1, a compound reported to increase the affinity of HDL for SR-BI, blocks SR-BI-mediated lipid transport (38.Nieland T.J. Penman M. Dori L. Krieger M. Kirchhausen T. Discovery of chemical inhibitors of the selective transfer of lipids mediated by the HDL receptor SR-BI.Proc. Natl. Acad. Sci. USA. 2002; 99: 15422-15427Crossref PubMed Scopus (180) Google Scholar). Together, these observations suggest that 1) binding of HDL to SR-BI is not sufficient for lipid transfer and 2) the HDL particle must be properly positioned for the formation of a "productive complex" to allow efficient lipid uptake to occur. Although there is some controversy over how SR-BI facilitates the transfer of HDL lipids to the cell, which may be attributable in part to cell type-specific differences in cholesterol metabolism, there is less disagreement over the fate of HDL CE after uptake has occurred. In fact, SR-BI has been shown to deliver HDL CE into a metabolically active membrane pool, where they are hydrolyzed by cell type-specific neutral CE hydrolases (39.Connelly M.A. Kellner-Weiber G. Rothblat G.H. Williams D.L. Scavenger receptor, class B, type I (SR-BI)-directed HDL-cholesteryl ester hydrolysis.J. Lipid Res. 2003; 44: 331-341Abstract Full Text Full Text PDF PubMed Scopus (46) Google Scholar). In conjunction with these activities, SR-BI 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 (40.de la Llera-Moya M. Rothblat G.H. Connelly M.A. Kellner-Weibel G. Sakar S.W. Phillips M.C. Williams D.L. Scavenger receptor BI (SR-BI) mediates free cholesterol flux independently of HDL tethering to the cell surface.J. Lipid Res. 1999; 40: 575-580Abstract Full Text Full Text PDF PubMed Google Scholar, 41.Kellner-Weibel G. de la Llera-Moya M. Connelly M.A. Stoudt G. Christian A.E. Haynes M.P. Williams D.L. Rothblat G.H. Expression of scavenger receptor BI in COS-7 cells alters cholesterol content and distribution.Biochemistry. 2000; 39: 221-229Crossref PubMed Scopus (133) Google Scholar). These data support the idea that SR-BI delivers HDL CE and FC into a metabolically active membrane pool where they are efficiently metabolized. In a recent publication, we reported that changing a glycine residue in the extracellular region of SR-BI near the C-terminal transmembrane domain to histidine had a profound effect on the ability of the receptor to deliver HDL CE in a way that allowed its efficient metabolism (42.Parathath S. Sahoo D. Darlington Y.F. Peng Y. Collins H.L. Rothblat G.H. Williams D.L. Connelly M.A. Glycine 420 near the C-terminal transmembrane domain of SR-BI is critical for proper delivery and metabolism of high density lipoprotein cholesteryl ester.J. Biol. Chem. 2004; 279: 24976-24985Abstract Full Text Full Text PDF PubMed Scopus (25) Google Scholar). Despite the fact that the mutant receptor, G420H, retained the ability to bind HDL particles, it exhibited a reduced ability to mediate selective HDL CE uptake and deliver HDL CE to a membrane pool, where it could be acted upon by a neutral CE hydrolase (42.Parathath S. Sahoo D. Darlington Y.F. Peng Y. Collins H.L. Rothblat G.H. Williams D.L. Connelly M.A. Glycine 420 near the C-terminal transmembrane domain of SR-BI is critical for proper delivery and metabolism of high density lipoprotein cholesteryl ester.J. Biol. Chem. 2004; 279: 24976-24985Abstract Full Text Full Text PDF PubMed Scopus (25) Google Scholar). This was evident by a decrease in the percentage of HDL CE that was hydrolyzed as well as by a lack of augmentation in the pool of membrane cholesterol that was sensitive to cholesterol oxidase. Interestingly, replacement of glycine 420 with an alanine, G420A, had no affect on receptor function (42.Parathath S. Sahoo D. Darlington Y.F. Peng Y. Collins H.L. Rothblat G.H. Williams D.L. Connelly M.A. Glycine 420 near the C-terminal transmembrane domain of SR-BI is critical for proper delivery and metabolism of high density lipoprotein cholesteryl ester.J. Biol. Chem. 2004; 279: 24976-24985Abstract Full Text Full Text PDF PubMed Scopus (25) Google Scholar). In this study, we examined the consequences of further amino acid substitution at glycine 420 to understand the disparity between the functional consequences of these two mutant receptors, G420H and G420A. The results revealed that substitution of glycine 420 with amino acids containing positively charged side chains disrupted SR-BI cholesterol transport function, whereas those containing large bulky side chains did not. In addition, substitution of glycine 420 with amino acids containing either a negatively charged side chain or a proline residue reduced receptor cell surface expression or disrupted the formation of a productive complex between SR-BI and HDL, respectively. These results, coupled with the fact that glycine 420 and the amino acid residues surrounding it are evolutionarily conserved, lend support to the supposition that this region of the receptor is critical for SR-BI-mediated CE and FC delivery and its subsequent metabolism. PCR amplifications were performed using a Perkin-Elmer Cetus DNA Thermal Cycler 9700 (Perkin-Elmer, Inc.). Oligonucleotides were purchased from Integrated DNA Technologies. The cloning procedure for pSG5(SR-BI), which contains the mouse SR-BI coding region, was described previously (43.Connelly M.A. Klein S.M. Azhar S. Abumrad N.A. Williams D.L. Comparison of class B scavenger receptors, CD36 and SR-BI, shows that both receptors mediate HDL-cholesteryl ester selective uptake but SR-BI exhibits a unique enhancement of cholesteryl ester uptake.J. Biol. Chem. 1999; 274: 41-47Abstract Full Text Full Text PDF PubMed Scopus (192) Google Scholar). The following primers were used to amplify pSG5(SR-BI) and introduce mutations into mouse SR-BI. Cloning of G420A and G420H was described previously (42.Parathath S. Sahoo D. Darlington Y.F. Peng Y. Collins H.L. Rothblat G.H. Williams D.L. Connelly M.A. Glycine 420 near the C-terminal transmembrane domain of SR-BI is critical for proper delivery and metabolism of high density lipoprotein cholesteryl ester.J. Biol. Chem. 2004; 279: 24976-24985Abstract Full Text Full Text PDF PubMed Scopus (25) Google Scholar). G420K, 5′-AGCCAGCTCTTCAAGCAAGGCAATGGGTGGCAAGCCCCTGAGCACG-3′ and 5′-AGCCAGCTCTTCAGCTCTGTTCGAACCACAGCAACGGCAGAAC-3′; G420E, 5′-AGCCAGCTCTTCAAGCGAAGCAATGGGTGGCAAGCCCCTGAGCACG-3′ and 5′-AGCCAGCTCTTCAGCTCTGTTCGAACCACAGCAACGGCAGAAC-3′; G420L, 5′-AGCCAGCTCTTCAAGCCTAGCAATGGGTGGCAAGCCCCTGAGCACG-3′ and 5′-AGCCAGCTCTTCAGCTCTGTTCGAACCACAGCAACGGCAGA-AC-3′; G420I, 5′-AGCCAGCTCTTCAAGCATAGCAATGGGTGGCAAGCCCCTGAGCACG-3′ and 5′-AGCCAGCTCTTCAGCTCTGTTCGAACCACAGCAACGGCAGAAC-3′; G420P, 5′-AGCCAGCTCTTCAAGCCCAGCAATGGGTGGCAAGCCCCTGAGCACG-3′ and 5′-AGCCAGCTCTTCAGCTCTGTTCGAACCACAGCAACGGCAGAAC-3′; G420Q, 5′-AGCCAGCTCTTCAAGCCAAGCAATGGGTGGCAAGCCCCTGAGCACG-3′ and 5′-AGCCAGCTCTTCAGCTCTGTTCGAACCACAGCAACGGCAGAAC-3′; G420F, 5′-AGCCAGCTCTTCAAGCTTCGCAATGGGTGGCAAGCCCCTGAGCACG-3′ and 5′-AGCCAGCTCTTCAGCTCTGTTCGAACCACAGCAACGGCAGAAC-3′. The resulting PCR products were digested with Sap I (New England Biolabs, Inc.) and recircularized. All plasmids were prepared using Endotoxin-free Qiagen Maxi-prep kits and sequenced throughout the SR-BI coding region to confirm the correct point mutation and to ensure that no undesired mutations had been generated during the amplification process. DNA sequencing was performed by the automated sequencing facility at Stony Brook University. Reactions were prepared using a dye termination cycle sequencing kit and analyzed on an Applied Biosystems model 3100 DNA Sequencer with an Excel Upgrade as recommended by the manufacturer (PE Applied Biosystems). COS-7 cells were maintained and transfected as described previously (43.Connelly M.A. Klein S.M. Azhar S. Abumrad N.A. Williams D.L. Comparison of class B scavenger receptors, CD36 and SR-BI, shows that both receptors mediate HDL-cholesteryl ester selective uptake but SR-BI exhibits a unique enhancement of cholesteryl ester uptake.J. Biol. Chem. 1999; 274: 41-47Abstract Full Text Full Text PDF PubMed Scopus (192) Google Scholar). The cells were assayed at 48 h after transfection unless indicated otherwise. Cell lysates were made (44.Rigotti A. Edelman E.R. Seifert P. Iqbal S.N. DeMattos R.B. Temel R.E. Krieger M. Williams D.L. Regulation by adrenocorticotropic hormone of the in vivo expression of scavenger receptor class B type I (SR-B1), a high density lipoprotein receptor, in steroidogenic cells of the murine adrenal gland.J. Biol. Chem. 1996; 271: 33545-33549Abstract Full Text Full Text PDF PubMed Scopus (204) Google Scholar, 45.Temel R.E. Trigatti B. DeMattos R.B. Azhar S. Krieger M. Williams D.L. Scavenger receptor B, type I (SR-BI) is the major route for the delivery of high density lipoprotein cholesterol to the steroidogenic pathway in cultured mouse adrenocortical cells.Proc. Natl. Acad. Sci. USA. 1997; 94: 13600-13605Crossref PubMed Scopus (209) Google Scholar), and protein concentrations were determined by the method of Lowry et al. (46.Lowry O.H. Rosebrough N.J. Farr A.L. Randall R.J. Protein measurement with the Folin phenol reagent.J. Biol. Chem. 1951; 193: 265-275Abstract Full Text PDF PubMed Google Scholar). Protein lysates were electrophoresed, transferred onto nitrocellulose membranes, and detected using a polyclonal anti-SR-BI C-terminal antibody (Novus Biologicals, Inc.) (1:5,000), a horseradish peroxidase-conjugated anti-rabbit secondary antibody (Jackson ImmunoResearch Laboratories) (1:10,000), and SuperSignal West Pico reagent (Pierce, Inc.). Human HDL3 (1.125 < d < 1.210 g/ml), herein referred to as HDL, was isolated by sequential ultracentrifugation (47.Havel R.J. Eder H.A. Bragdon J.H. The distribution and chemical composition of ultracentrifugally separated lipoproteins in human serum.J. Clin. Invest. 1955; 34: 1345-1353Crossref PubMed Scopus (6480) Google Scholar). The HDL was labeled with either nonhydrolyzable [3H]cholesteryl oleyl ether ([3H]COE) (Amersham Life Sciences) or hydrolyzable [3H]cholesteryl oleate/[3H]CE (Amersham Life Sciences) using recombinant CE transfer protein (Cardiovascular Targets, Inc.) as described (48.Francone O.L. Haghpassand M. Bennett J.A. Royer L. McNeish J. Expression of human lecithin:cholesterol acyltransferase in transgenic mice: effects on cholesterol efflux, esterification, and transport.J. Lipid Res. 1997; 38: 813-822Abstract Full Text PDF PubMed Google Scholar) with modifications (49.Connelly M.A. de la Llera-Moya M. Peng Y. Drazul-Schrader D. Rothblat G.H. Williams D.L. Separation of lipid transport functions by mutations in the extracellular domain of scavenger receptor class B, type I (SR-BI).J. Biol. Chem. 2003; 279: 24976-24985Google Scholar). Labeled particles were reisolated by gel exclusion chromatography and then labeled with 125I-dilactitol tyramine as described previously (43.Connelly M.A. Klein S.M. Azhar S. Abumrad N.A. Williams D.L. Comparison of class B scavenger receptors, CD36 and SR-BI, shows that both receptors mediate HDL-cholesteryl ester selective uptake but SR-BI exhibits a unique enhancement of cholesteryl ester uptake.J. Biol. Chem. 1999; 274: 41-47Abstract Full Text Full Text PDF PubMed Scopus (192) Google Scholar). The average specific activity of the 125I-dilactitol tyramine-[3H]COE-HDL was 330 dpm/ng protein for 125I and 4.0 dpm/ng protein (16.7 dpm/ng CE) for 3H. The average specific activity of the [3H]CE-HDL was 13.5 dpm/ng protein (52.1 dpm/ng CE) for 3H. Transiently transfected COS-7 cells (in 35 mm wells) were washed once with serum-free DMEM and 0.5% BSA, and 125I-dilactitol tyramine-[3H]COE-HDL particles were added at a concentration of 10 μg protein/ml (unless indicated otherwise) in serum-free DMEM 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 acid-soluble 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 (43.Connelly M.A. Klein S.M. Azhar S. Abumrad N.A. Williams D.L. Comparison of class B scavenger receptors, CD36 and SR-BI, shows that both receptors mediate HDL-cholesteryl ester selective uptake but SR-BI exhibits a unique enhancement of cholesteryl ester uptake.J. Biol. Chem. 1999; 274: 41-47Abstract Full Text Full Text PDF PubMed Scopus (192) Google Scholar). The efficiency of HDL CE selective uptake was determined by subtracting the values for vector-transfected cells and normalizing the amount of HDL CE selective uptake to the amount of cell-associated HDL particles. Statistical comparisons were made by one-way ANOVA with Bonferroni posttest for all groups (* P < 0.05, ** P < 0.001) compared with wild-type SR-BI (GraphPad Prism version 4.0 software). Transiently transfected COS-7 cells (in 35 mm wells) were washed with cold PBS (with calcium and magnesium) and incubated for 1 h at 4°C in the presence of biotin (1 mg/ml). Cells were washed with PBS containing 100 mM glycine and lysed in RIPA buffer (50 mM Tris, pH 7.4, 150 mM NaCl, 1% Nonidet P-40, 0.5% sodium deoxycholate, and 0.1% sodium dodecyl sulfate) containing Halt Protease Inhibitor Cocktail (Pierce, Inc.). After centrifugation, a portion of the clarified lysate was incubated with Neutravidin beads (Pierce, Inc.) for 1 h at room temperature. The beads were pelleted, washed four times with 1 ml of RIPA buffer per wash, and resuspended in 50–200 μl of gel loading buffer. Aliquots of total lysate and surface proteins, equivalent to the original amount of total lysate that was precipitated by the Neutravidin beads, were electrophoresed on 10% polyacrylamide gels, transferred onto nitrocellulose membranes, and detected using a polyclonal anti-SR-BI C-terminal antibody as described above. Assays for cholesterol influx (4 h of incubation with 10 μg/ml HDL3 as the donor particles) (49.Connelly M.A. de la Llera-Moya M. Peng Y. Drazul-Schrader D. Rothblat G.H. Williams D.L. Separation of lipid transport functions by mutations in the extracellular domain of scavenger receptor class B, type I (SR-BI).J. Biol. Chem. 2003; 279: 24976-24985Google Scholar), cholesterol efflux (4 h of incubation with 50 μg/ml HDL3 as the acceptor particles), and CE hydrolysis (2 h of incubation with 10 μg/ml HDL3) (42.Parathath S. Sahoo D. Darlington Y.F. Peng Y. Collins H.L. Rothblat G.H. Williams D.L. Connelly M.A. Glyci
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