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Dissociation of the High Density Lipoprotein and Low Density Lipoprotein Binding Activities of Murine Scavenger Receptor Class B Type I (mSR-BI) Using Retrovirus Library-based Activity Dissection

2000; Elsevier BV; Volume: 275; Issue: 13 Linguagem: Inglês

10.1074/jbc.275.13.9120

ISSN

1083-351X

Autores

Xiang-ju Gu, Roger Lawrence, Monty Krieger,

Tópico(s)

Lipoproteins and Cardiovascular Health

Resumo

The murine class B, type I scavenger receptor (mSR-BI) is a receptor for both high density lipoprotein (HDL) and low density lipoprotein (LDL) and mediates selective, rather than endocytic, uptake of lipoprotein lipid. We have developed a "retrovirus library-based activity dissection" method to generate mSR-BI mutants in which some, but not all, of the activities of this multifunctional protein have been disrupted. This method employs three techniques: 1) efficient in vitro cDNA mutagenesis (here error-prone PCR was used), 2) efficient retroviral delivery and high expression of single mutant cDNAs into individual cells, and 3) isolation of infected cells expressing the desired mutant phenotype using high sensitivity positive/negative screening by two-color fluorescence-activated cell sorting. A set of mutants, all having arginine substitutions at two common sites (positions 402 or 401 and position 418), were isolated and characterized. Mutation at either site alone did not generate as strong a mutant phenotype (loss of DiI uptake from DiI-HDL) as did the double mutations. "Activity-dissected" double mutants were as effective as wild-type mSR-BI in functioning as LDL receptors, mediating high affinity LDL binding and uptake of metabolically active cholesterol from LDL, but they lost most of their corresponding HDL receptor activity. Thus, these mutants provide support for the proposal that the interaction of SR-BI with HDL differs from that with LDL. Examination of the in vivo function of such mutants may provide insights into the differential roles of the LDL and HDL receptor activities of SR-BI in normal lipoprotein metabolism and in SR-BI's ability to protect against atherosclerosis. The murine class B, type I scavenger receptor (mSR-BI) is a receptor for both high density lipoprotein (HDL) and low density lipoprotein (LDL) and mediates selective, rather than endocytic, uptake of lipoprotein lipid. We have developed a "retrovirus library-based activity dissection" method to generate mSR-BI mutants in which some, but not all, of the activities of this multifunctional protein have been disrupted. This method employs three techniques: 1) efficient in vitro cDNA mutagenesis (here error-prone PCR was used), 2) efficient retroviral delivery and high expression of single mutant cDNAs into individual cells, and 3) isolation of infected cells expressing the desired mutant phenotype using high sensitivity positive/negative screening by two-color fluorescence-activated cell sorting. A set of mutants, all having arginine substitutions at two common sites (positions 402 or 401 and position 418), were isolated and characterized. Mutation at either site alone did not generate as strong a mutant phenotype (loss of DiI uptake from DiI-HDL) as did the double mutations. "Activity-dissected" double mutants were as effective as wild-type mSR-BI in functioning as LDL receptors, mediating high affinity LDL binding and uptake of metabolically active cholesterol from LDL, but they lost most of their corresponding HDL receptor activity. Thus, these mutants provide support for the proposal that the interaction of SR-BI with HDL differs from that with LDL. Examination of the in vivo function of such mutants may provide insights into the differential roles of the LDL and HDL receptor activities of SR-BI in normal lipoprotein metabolism and in SR-BI's ability to protect against atherosclerosis. scavenger receptor class B type I high density lipoprotein low density lipoprotein very low density lipoprotein bovine serum albumin fluorescence-activated cell sorting 1,1′-dioctadecyl-3,3,3′,3′-tetramethylindocarbocyanine perchlorate phosphate-buffered saline Chinese hamster ovary polymerase chain reaction kilobase(s) Dulbecco's modified Eagle's medium placental alkaline phosphatase multiplicity of infection acetyl-CoA:cholesterol acyltransferase Scavenger receptors (1.Acton S.L. Scherer P.E. Lodish H.F. Krieger M. J. Biol. Chem. 1994; 269: 21003-21009Abstract Full Text PDF PubMed Google Scholar, 2.Krieger M Annu. Rev. Biochem. 1999; 68: 523-558Crossref PubMed Scopus (458) Google Scholar) are multifunctional proteins defined by their ability to bind chemically modified lipoproteins (3.Krieger M. Herz J. Annu. Rev. Biochem. 1994; 63: 601-637Crossref PubMed Scopus (1058) Google Scholar). Often scavenger receptors can mediate the binding of a wide array of other types macromolecules or macromolecular complexes (3.Krieger M. Herz J. Annu. Rev. Biochem. 1994; 63: 601-637Crossref PubMed Scopus (1058) Google Scholar, 4.Krieger M. Curr. Opin. Lipidol. 1997; 8: 275-280Crossref PubMed Scopus (253) Google Scholar, 5.Greaves D.R. Gough P.J. Gordon S. Curr. Opin. Lipidol. 1998; 9: 425-432Crossref PubMed Scopus (104) Google Scholar). For example, scavenger receptor class B, type I, SR-BI1 (1.Acton S.L. Scherer P.E. Lodish H.F. Krieger M. J. Biol. Chem. 1994; 269: 21003-21009Abstract Full Text PDF PubMed Google Scholar, 2.Krieger M Annu. Rev. Biochem. 1999; 68: 523-558Crossref PubMed Scopus (458) Google Scholar), which was the first molecularly well defined native HDL receptor identified (6.Acton S. Rigotti A. Landschulz K.T. Xu S. Hobbs H.H. Krieger M. Science. 1996; 271: 518-520Crossref PubMed Scopus (1988) Google Scholar), can also bind unmodified LDL, unmodified VLDL, anionic phospholipids, as well as a number of modified proteins including acetylated LDL, oxidized LDL, and maleylated-BSA (1.Acton S.L. Scherer P.E. Lodish H.F. Krieger M. J. Biol. Chem. 1994; 269: 21003-21009Abstract Full Text PDF PubMed Google Scholar, 7.Rigotti A. Acton S.L. Krieger M. J. Biol. Chem. 1995; 270: 16221-16224Abstract Full Text Full Text PDF PubMed Scopus (489) Google Scholar, 8.Calvo D. Gomez-Coronado D. Lasuncion M.A. Vega M.A. Arterioscler. Thromb. Vasc. Biol. 1997; 17: 2341-2349Crossref PubMed Scopus (211) Google Scholar). In transfected cultured cells expressing high levels of murine (m)SR-BI, HDL is a very effective competitor for mSR-BI-mediated 125I-HDL cell association (6.Acton S. Rigotti A. Landschulz K.T. Xu S. Hobbs H.H. Krieger M. Science. 1996; 271: 518-520Crossref PubMed Scopus (1988) Google Scholar). In contrast, LDL, which binds more tightly to mSR-BI than HDL (1.Acton S.L. Scherer P.E. Lodish H.F. Krieger M. J. Biol. Chem. 1994; 269: 21003-21009Abstract Full Text PDF PubMed Google Scholar, 6.Acton S. Rigotti A. Landschulz K.T. Xu S. Hobbs H.H. Krieger M. Science. 1996; 271: 518-520Crossref PubMed Scopus (1988) Google Scholar), only poorly competes for HDL association (Ref. 6.Acton S. Rigotti A. Landschulz K.T. Xu S. Hobbs H.H. Krieger M. Science. 1996; 271: 518-520Crossref PubMed Scopus (1988) Google Scholar, and see below). These findings suggested distinct modes of binding, and perhaps distinct binding sites, for these lipoproteins on mSR-BI. Unlike the classic LDL receptor, which mediates endocytosis of the intact LDL particle via coated pits and vesicles and its subsequent hydrolysis in lysosomes (9.Goldstein J.L. Brown M.S. Anderson R.G. Russell D.W. Schneider W.J. Annu. Rev. Cell Biol. 1985; 1: 1-39Crossref PubMed Scopus (1106) Google Scholar), SR-BI mediates the selective uptake of the cholesteryl esters from HDL (6.Acton S. Rigotti A. Landschulz K.T. Xu S. Hobbs H.H. Krieger M. Science. 1996; 271: 518-520Crossref PubMed Scopus (1988) Google Scholar). Selective lipid uptake is fundamentally different from LDL receptor-mediated endocytosis. It involves efficient transfer of the lipid, but not the protein (apolipoprotein), components from HDL to cells, and it does not involve the internalization and subsequent degradation of the intact lipoprotein particle (10, 11, reviewed in Ref. 2.Krieger M Annu. Rev. Biochem. 1999; 68: 523-558Crossref PubMed Scopus (458) Google Scholar). Recent studies suggest that SR-BI-mediated selective lipid uptake is a two-step process, in which high affinity lipoprotein binding is followed by receptor-mediated transfer of lipid from the lipoprotein particle to the cell membrane (12.Gu 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 (199) Google Scholar, 13.Connelly 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 (192) Google Scholar). After lipid transfer, the lipid-depleted lipoprotein particle is released from the cells and re-enters the extracellular space. SR-BI can mediate selective uptake of lipid from LDL as well as HDL (14.Stangl H. Cao G. Wyne K.L. Hobbs H.H. J. Biol. Chem. 1998; 273: 31002-31008Abstract Full Text Full Text PDF PubMed Scopus (106) Google Scholar, 15.Swarnakar S. Temel R.E. Connelly M.A. Azhar S. Williams D.L. J. Biol. Chem. 1999; 274: 29733-29739Abstract Full Text Full Text PDF PubMed Scopus (114) Google Scholar, 16.Stangl H. Hyatt M. Hobbs H. J. Biol. Chem. 1999; 274: 32692-32698Abstract Full Text Full Text PDF PubMed Scopus (124) Google Scholar). The detailed molecular mechanism(s) underlying selective uptake have not yet been elucidated. SR-BI is highly expressed in liver and steroidogenic tissues and plays a central role in controlling plasma HDL levels and cholesterol stores in steroidogenic tissues (2.Krieger M Annu. Rev. Biochem. 1999; 68: 523-558Crossref PubMed Scopus (458) Google Scholar, 6.Acton S. Rigotti A. Landschulz K.T. Xu S. Hobbs H.H. Krieger M. Science. 1996; 271: 518-520Crossref PubMed Scopus (1988) Google Scholar, 17.Rigotti 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 (749) Google Scholar). Liver-specific overexpression of SR-BI in mice can result in decreases in cholesterol in VLDL, LDL/intermediate density lipoprotein, and HDL and reductions in apoB and apoA-I (18.Kozarsky K.F. Donahee M.H. Rigotti A. Iqbal S.N. Edelman E.R. Krieger M. Nature. 1997; 387: 414-417Crossref PubMed Scopus (626) Google Scholar, 19.Wang N. Arai T. Ji Y. Rinninger F. Tall A.R. J. Biol. Chem. 1998; 273: 32920-32926Abstract Full Text Full Text PDF PubMed Scopus (244) Google Scholar, 20.Ueda Y. Royer L. Gong E. Zhang J. Cooper P.N. Francone O. Rubin E.M. J. Biol. Chem. 1999; 274: 7165-7171Abstract Full Text Full Text PDF PubMed Scopus (214) Google Scholar). Furthermore, SR-BI overexpression has been reported to reduce diet (high fat, high cholesterol) induced increases in VLDL and LDL apoB in transgenic mice (19.Wang N. Arai T. Ji Y. Rinninger F. Tall A.R. J. Biol. Chem. 1998; 273: 32920-32926Abstract Full Text Full Text PDF PubMed Scopus (244) Google Scholar, 20.Ueda Y. Royer L. Gong E. Zhang J. Cooper P.N. Francone O. Rubin E.M. J. Biol. Chem. 1999; 274: 7165-7171Abstract Full Text Full Text PDF PubMed Scopus (214) Google Scholar). These data suggest that SR-BI might play a role in LDL as well as HDL metabolism in vivo. An SR-BI null mutation in the chow-diet fed apoE KO mouse model of atherosclerosis dramatically accelerates atherosclerosis (21.Trigatti B. Rayburn H. Vinals M. Braun A. Miettinen H. Penman M. Hertz M. Schrenzel M. Amigo L. Rigotti A. Krieger M. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 9322-9327Crossref PubMed Scopus (437) Google Scholar), and overexpression of SR-BI in either fat-fed LDL receptor-deficient or apoE-deficient mice decreases atherosclerosis (22.Arai T. Wang N. Bezouevski M. Welch C. Tall A.R. J. Biol. Chem. 1999; 274: 2366-2371Abstract Full Text Full Text PDF PubMed Scopus (284) Google Scholar, 23.Chase M.B. Santamarina-Fojo S. Shamburek R.D. Amar M.C. Knapper C.L. Meyn S.M. Brewer Jr., H.B. Circulation. 1998; 98: I-202Google Scholar, 24.Kozarsky K.F. Donahee M.H. Glick J.M. Krieger M. Rader D.J. Arterioscler. Thromb. Vasc. Biol. 2000; 20: 721-727Crossref PubMed Scopus (305) Google Scholar). Although these studies show that SR-BI is atheroprotective in mice, and thus a potential therapeutic target in humans, the relative importance of the activities of SR-BI as a receptor for HDLversus LDL for this protection from atherosclerosis remains uncertain (22.Arai T. Wang N. Bezouevski M. Welch C. Tall A.R. J. Biol. Chem. 1999; 274: 2366-2371Abstract Full Text Full Text PDF PubMed Scopus (284) Google Scholar, 24.Kozarsky K.F. Donahee M.H. Glick J.M. Krieger M. Rader D.J. Arterioscler. Thromb. Vasc. Biol. 2000; 20: 721-727Crossref PubMed Scopus (305) Google Scholar). Structure/function analysis of multifunctional proteins such as SR-BI can be facilitated by the identification of mutant forms of those proteins in which a subset of their activities or properties are disrupted. Here we describe a method to isolate such mutants, called retrovirus library-based activity dissection. This approach is based on three techniques: 1) efficient mutagenesis of all or part of the cDNA encoding the multifunctional protein to generate a large, complex library of mutant cDNAs (here error-prone PCR was used); 2) efficient delivery of single mutant cDNAs from the library into individual cells and high level expression using retroviruses and retrovirus-receptor expressing cells; and 3) isolation of infected cells expressing the desired mutant phenotype (retention of some functions, loss of others) using high sensitivity positive/negative screening by two-color fluorescence-activated cell sorting (FACS). Others have previously reported the use of random mutagenesis, retroviral cDNA transduction, and selection to isolate gain of function mutants with a single mutant phenotype (e.g.altered cell morphology or hormone-independent cell growth, see Refs.25.Kimelman D. Mol. Cell. Biol. 1986; 6: 1487-1496Crossref PubMed Scopus (6) Google Scholar and 26.Onishi M. Nosaka T. Misawa K. Mui A.L.-F. Gorman D. McMahon M. Miyajima A. Kitamura T. Mol. Cell. Biol. 1998; 18: 3871-3879Crossref PubMed Scopus (347) Google Scholar). Using the retrovirus library-based activity dissection method, we identified two sites in mSR-BI that, when simultaneously mutated to arginines, dramatically reduced native HDL binding to the receptor without substantially altering the levels of high affinity LDL binding or uptake of metabolically active cholesterol from LDL. Such "activity dissected" mutants should help clarify the molecular basis for the complex binding properties of SR-BI and, if expressed in animal models, may help provide insights into the mechanism(s) underlying the atheroprotective effects of SR-BI. Human HDL, LDL, 125I-labeled HDL (125I-HDL), 125I-labeled LDL (125I-LDL), and DiI (1,1′-dioctadecyl-3,3,3′,3′-tetramethylindocarbocyanine perchlorate)-labeled HDL (DiI-HDL) were prepared as described previously (6.Acton S. Rigotti A. Landschulz K.T. Xu S. Hobbs H.H. Krieger M. Science. 1996; 271: 518-520Crossref PubMed Scopus (1988) Google Scholar, 12.Gu 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 (199) Google Scholar). All 125I-HDL preparations were monitored by SDS-polyacrylamide gel electrophoresis to ensure that the preparations were free of radiolytic or oxidative damage. Alexa 488 was purchased from Molecular Probes (Eugene, OR). Alexa 488-labeled HDL (Alexa-HDL) was prepared following the manufacturer's suggestions. In brief, 50 μl of 1 m bicarbonate were added to 0.5 ml of 2 mg of protein/ml HDL in PBS and the mixture was then transferred to a vial of reactive dye (Alexa 488 carboxylic acid, succinimidyl ester, dilithium salt). The reaction mixture was stirred for 1 h at room temperature before the reaction was stopped by adding 15 μl of hydroxylamine. The labeled HDL was separated from the free dye by exclusion chromatography using the column supplied by the manufacturer. Based on SDS-polyacrylamide gel electrophoresis, Alexa 488, which was expected to react with primary amines of proteins (Molecular Probes, Alexa 488 Protein Labeling Kit Manual, catalog number A-10235), primarily covalently labeled the two major apolipoproteins on the surface of HDL, apoA-I and apoA-II (27.Assman G. von Eckardstein A. Brewer H.B. Scriver C.R. Beaudet A.L. Sly W.S. Valle D. The Metabolic and Molecular Bases of Inherited Disease. McGraw-Hill, New York1995: 2053-2072Google Scholar, 28.Zannis V.I. Kardassis D. Zanni E.E. Adv. Hum. Genet. 1993; 21: 145-319Crossref PubMed Scopus (89) Google Scholar) (data not shown). In contrast, in DiI-HDL the DiI associated with the HDL particles but had not covalently labeled the apolipoproteins (not shown). The human CD36 (hCD36) expression vector (29.Oquendo P. Hundt E. Lawler J. Seed B. Cell. 1989; 58: 95-101Abstract Full Text PDF PubMed Scopus (400) Google Scholar) was the generous gift of B. Seed (Massachusetts General Hospital, Boston). All of the expression vectors for mSR-BI were based on pCDNA1 (Invitrogen). Wild-type mSR-BI expression vectors used for these studies included pmSR-BI number 77 (6.Acton S. Rigotti A. Landschulz K.T. Xu S. Hobbs H.H. Krieger M. Science. 1996; 271: 518-520Crossref PubMed Scopus (1988) Google Scholar) and minor variants (e.g. ex47 and ex68) 2X. Gu, R. Lawrence, and M. Krieger, unpublished data. involving small differences in the linker region to facilitate cloning and restriction digestion. CHO cells were grown or incubated in medium A (Ham's F-12 containing 50 units/ml of penicillin, 50 μg/ml streptomycin, and 2 mm glutamine) supplemented with serum or other additions as indicated. PCR-based random mutagenesis was performed under two different conditions according to the protocols by Leunget al. (30.Leung D. Chen E. Goeddel D. Technique. 1989; 1: 11-15Google Scholar) and Cadwell et al. (31.Cadwell C.R. Joyce G.F. PCR Methods Appl. 1992; 2: 28-33Crossref PubMed Scopus (827) Google Scholar) with some modifications. Under the first condition (30.Leung D. Chen E. Goeddel D. Technique. 1989; 1: 11-15Google Scholar), the reaction mixture contained 1 mm dGTP, 1 mm dCTP, 1 mm dTTP, 0.2 mm dATP, 0.5 mmMnCl2, and 5.5 mm MgCl2. pmSR-BI number 77 (6.Acton S. Rigotti A. Landschulz K.T. Xu S. Hobbs H.H. Krieger M. Science. 1996; 271: 518-520Crossref PubMed Scopus (1988) Google Scholar) containing the wild-type mSR-BI was used as the template. Primers XG65 (5′-GGAAGATCTCCGTCTCCTTCAGGTCCTGAGC-3′) and XG66 (5′-TTATCGTCGACGCGTGGGCATCCATGTGCCGT-3′) were used at a concentration of 500 pmol/ml. Under the second condition, 0.2 mm dGTP was used in order to reduce the bias for A/T > G/C change (31.Cadwell C.R. Joyce G.F. PCR Methods Appl. 1992; 2: 28-33Crossref PubMed Scopus (827) Google Scholar). Amplification was performed on a Perkin-Elmer DNA cycler: initial denaturation (94 °C for 4 min) followed by 40 cycles of annealing (50 °C for 1 min), elongation (72 °C for 2 min), and denaturation (94 °C for 1 min). The PCR products from 25 replicate tubes of each PCR under both conditions were pooled together to generate the collection of mutated 1.7-kb products used to construct the mutant library. Plasmid pMX-mSR-BI was generated by blunt-end ligating the HindIII/XbaI fragment of pmSR-BI′ (12.Gu 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 (199) Google Scholar) which contains the coding sequence of the wild-type mSR-BI into theNotI/EcoRI site of the pMX vector (32.Liu X. Sun Y. Constantinescu S.N. Karam E. Weinberg R.A. Lodish H.F. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 10669-10674Crossref PubMed Scopus (331) Google Scholar). For generating the Sub4 mutant library, the 1.7-kb fragments generated by error-prone PCR were digested with BlpI/SalI and cloned into the corresponding position in pMX-mSR-BI. Plasmids were isolated from pooled Escherichia coli colonies without further amplification. Retroviruses were generated in the Phoenix packaging cell line (ATCC SD 3444, grown in DMEM containing 10% fetal bovine serum, 50 units/ml penicillin, 50 μg/ml streptomycin, 2 mm glutamine) provided by G. Nolan (Stanford Medical Center) by transfecting the cells with the pMX-mSR-BI plasmid or the Sub4 plasmid library of the mSR-BI mutants using the calcium phosphate method (33.Chatterton J.E. Hirsch D. Schwartz J.J. Bickel P.E. Rosenberg R.D. Lodish H.F. Krieger M. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 915-920Crossref PubMed Scopus (52) Google Scholar). Supernatants were collected 24 and 48 h after transfection, and either were used immediately to infect CHO[Eco] cells (see below) or were frozen in liquid nitrogen and stored at −80 °C before use. Stable CHO[Eco] cell lines expressing the ecotropic retrovirus receptor (34.Albritton L.M. Tseng L. Scadden D. Cunningham J.M. Cell. 1989; 57: 659-666Abstract Full Text PDF PubMed Scopus (553) Google Scholar) were generated by transfecting CHO cells with the plasmid pCB7-Eco (gift from H. Lodish, Whitehead Institute, Cambridge, MA, Ref. 33.Chatterton J.E. Hirsch D. Schwartz J.J. Bickel P.E. Rosenberg R.D. Lodish H.F. Krieger M. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 915-920Crossref PubMed Scopus (52) Google Scholar) using LipofectAMINE (Life Technologies, Grand Island, NY). Cells were selected in medium A containing 5% fetal bovine serum (medium B) and 250 μg/ml hygromycin B. Individual colonies were then tested for the expression of human placental alkaline phosphatase (PLAP) after infection with a PLAP-expressing retrovirus construct (MSCV-PLAP, gift from D. Baltimore, Caltech, Pasadena, CA) and the clone which gave the highest infection rate was isolated. This clone, designated CHO[Eco], was used for subsequent studies to express retrovirally encoded mSR-BI proteins. For transduction, CHO[Eco] cells were set at 500,000 cells/100-mm dish in medium B on day 0. On day 1, the cells were transduced with 6 ml of medium B containing 6 μg/ml of the adjuvent Polybrene and retrovirus supernatants diluted (1:3 or 1:6) in this medium. After incubation for 24 h at 34 °C, the retrovirus containing medium was replaced with fresh medium B. On day 3, cells were harvested with trypsin, reset into two 100-mm dishes, and maintained in medium B without selection. On day 5, cells were processed for labeling with fluorescent lipoproteins and FACS (see below) or were harvested and stored frozen at −80 °C for future FACS screening. Cells transduced with retrovirus (see above) were washed twice with Ca2+- and Mg2+-free PBS and were then incubated at 37 °C for 2 h with DiI-HDL (5 μg of protein/ml) in medium A containing 0.5% (w/v) fatty acid free BSA (medium C). Cells were harvested with trypsin, the trypsin was quenched with medium B, and the cells were then washed once with medium C. Cells were then incubated at room temperature for 1 h with 10 μg of protein/ml Alexa-HDL in Ca2+- and Mg2+-free PBS containing 0.5% BSA. Immediately before FACS analysis and sorting, the cells were pelleted at 500 × g for 2 min, and resuspended in Ca2+- and Mg2+-free PBS containing 0.5% BSA. FACS analysis was performed with a Becton-Dickson FACStar instrument. ldlA[mSR-BI] cells (6.Acton S. Rigotti A. Landschulz K.T. Xu S. Hobbs H.H. Krieger M. Science. 1996; 271: 518-520Crossref PubMed Scopus (1988) Google Scholar) labeled separately with either DiI-HDL or Alexa-HDL were used to set the compensation of the instrument to correct the fluorescence signal spill-over between optical channels. Doubly labeled CHO[Eco] cell pools transduced with MX-mSR-BI virus at low multiplicity of infection (m.o.i.) were used as a reference to set the region for collecting the DiI−/Alexa+cells. Cells showing low DiI/Alexa signal ratio (DiI−/Alexa+) were isolated, expanded, and subjected to additional rounds of fluorescence labeling, FACS analysis, and sorting. Genomic DNAs from the DiI−/Alexa+ CHO[Eco] cells were isolated (35.Sambrook J. Fritsch E.F. Maniatis T. Molecular Cloning: A Laboratory Mannual. 2nd Ed. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY1989: 9.16-9.22Google Scholar) and the mutant mSR-BI transgenes were cloned using high fidelity Platinum Taq DNA polymerase (Life Technology) and primers MX1(5′-CCACCGCCCTCAAAGTAGACG-3′, upstream primer in the 5′-viral long terminal repeat region) and XG66 (downstream primer). The resulting 1.7-kb PCR fragments were digested withBglII/BstXI and cloned into theBamHI/BstXI sites in plasmid ex47. ThePstI/XbaI fragment of plasmid pmSR-BI number 77 (6.Acton S. Rigotti A. Landschulz K.T. Xu S. Hobbs H.H. Krieger M. Science. 1996; 271: 518-520Crossref PubMed Scopus (1988) Google Scholar) was cloned into the PstI/XbaI site of pUC18 to generate plasmid VM1. To generate point mutations at amino acid position 418 (glutamine in the wild-type sequence), we amplified PCR fragments using wild-type mSR-BI as template and primers: P5 (5′-GTTCCTTCACAAAGATCCTC-3′) and XG-71(Q/K) (5′-CTGTGGTTCGAAAAGAGCGGAGCAATGGGTGGC-3′); P5 and XG-72(Q/E) (5′-CTGTGGTTCGAAGAGAGCGGAGCAATGGGTGGC-3′); P5 and XG-73(Q/A) (5′-CTGTGGTTCGAAGCTAGCGGAGCAATGGGTGGC-3′); P5 and XG-74(Q/L) (5′-CTGTGGTTCGAACTGAGCGGAGCAATGGGTGGC-3′). The amplified fragments were digested with SfuI and XbaI and cloned into theSfuI/XbaI site of plasmid VM1 to generate plasmids VM15, VM16, VM17, and VM18 respectively. TheBlpI/XbaI fragments from these four plasmids were then cloned into the corresponding sites of pmSR-BI number 77 to generate plasmids VM23 (Q418K), VM24 (Q418E), VM25 (Q418A), and VM26 (Q418L), each encodes a mSR-BI mutant with a single amino acid substitution at Gln418. To generate point mutations at amino acid position 402 (glutamine in the wild-type sequence), we amplified PCR fragments using wild-type mSR-BI as template and primers: P5 and XG-67(Q/K) (5′-ATGCAGCTGAGCCTCTACATCAAATCTGTCAAGGGCATCGGGAAGACAGGGAAGATCGAGCCAG-3′); P5 and XG-68(Q/E) (5′-ATGCAGCTGAGCCTCTACATCAAATCTGTCAAGGGCATCGGGGAGACGGGGAAGATCGAGCCAGTAG-3′); P5 and XG-69(Q/A) (5′-ATGCAGCTGAGCCTCTACATCAAATCTGTCAAGGGCATCGGGGCTACCGGGAAGATCGAGCCAGTAG-3′); P5 and XG-70(Q/L) (5′-ATGCAGCTGAGCCTCTACATCAAATCTGTCAAGGGCATCGGGTTAACCGGGAAGATCGAGCCAGTAG-3′). The amplified fragments were digested with BlpI andXbaI and cloned into the corresponding site in pmSR-BI number 77 to generate VM19 (Q402K), VM22 (Q402E), VM20 (Q402A), and VM21 (Q402L), each encodes a mSR-BI mutant with a single amino acid substitution at Gln402. To generate the double mutant with Q402R/Q418R, we cloned theXbaI/FspI fragment from plasmid pmSR-BI number 77 into the corresponding site of plasmid M4-D (see "Results") to generate plasmid VM12′. The XbaI/BlpI fragment from VM12′ was then cloned into the corresponding site in plasmid ex68 to generate plasmid VM54, which encodes the mSR-BI mutant with the Q402R/Q418R double mutation. All the site-specific mutations mentioned above were confirmed by DNA sequencing. Cells grown in 6-well dishes were labeled with 1 ml of Ca2+- and Mg2+-free PBS containing 0.5% BSA and rabbit polyclonal antibody against the extracellular domain of mSR-BI (1:1000 dilution, generous gift from K. Kozarsky) for 1 h at room temperature. Cells were then washed 3 times with Ca2+- and Mg2+-free PBS and labeled with FITC-conjugated goat anti-rabbit IgG (1:1000 dilution, Cappel, West Chester, PA) in 1 ml of Ca2+- and Mg2+-free PBS containing 0.5% BSA, 2 mm EDTA. After 20 min, cells were detached from the plastic by gentle pipetting and incubated at room temperature for another 20 min. Cells were then pelleted and resuspended in 1 ml of Ca2+- and Mg2+-free PBS containing 0.5% BSA and subjected to fluorescence flow cytometric analysis. The mean value of the fluorescence intensity was used as a measure of mSR-BI surface expression. Cells were maintained in medium A containing 5% newborn calf lipoprotein-deficient serum (NC-LPDS, medium D) for at least 2 weeks before the ACAT assay to prevent differential buildup of intracellular cholesterol in cells expressing different lipoprotein receptors. Cells were set in 6-well dishes at 80,000 cell/well on day 0 in medium D and refed with the same medium on day 2. On day 3, cells were washed twice with Ca2+- and Mg2+-free PBS and refed with 1 ml of medium C containing the indicated amounts of either HDL or LDL. After a 5-h incubation at 37 °C, 10 μl (100 nmol) of [3H]oleate/BSA mixture (21,804 dpm/nmol, a gift from Dr. L. Liscum, Tufts University, Ref. 36.Goldstein J.L. Basu S.K. Brown M.S. Methods Enzymol. 1983; 98: 241-260Crossref PubMed Scopus (1278) Google Scholar) was added to each well. After incubation at 37 °C for 1.5 h, the cells were washed twice with ice-cold Tris wash buffer (50 mm Tris-HCl, 0.15m NaCl, pH 7.4) containing 2 mg/ml BSA, followed by two quick washes with Tris wash buffer without BSA. Two ml of hexane/isopropyl alcohol (3:2) were then added to each well. After 15 min of shaking at room temperature, 10 μl of recovery standard (2 mg/ml cholesterol oleate, 1 mg/ml triolein, 50 μCi/ml cholesterol [14C]oleate in chloroform/methanol (2:1)) were added to each well. The plates were shaken for another 15 min before the organic extracts were transferred to 12 × 75-mm glass tubes. Cells were extracted with addition of another 1 ml of hexane/isopropyl alcohol and the extracts were pooled and dried under nitrogen gas. Samples were then dissolved in 50 μl of chloroform and separated by silica gel thin layer chromatography (developed with heptane/diethyl ether/glacial acetic acid (90:30:1)). The cholesteryl esters were visualized by staining with iodine vapor and cut away from the silica gel plates (Silica Gel 60, Fisher Scientific). The amounts of cholesteryl [3H]oleate formed were measured using a Tri-carb liquid scintillation analyzer (Packard). For protein determinations, cells were dissolved in 1 ml of 0.1 n NaOH after solvent extraction and the protein levels determined using the method of Lowryet al. (37.Lowry O.H. Rosebrough N.J. Farr A.L. Randall R.J. J. Biol. Chem. 1951; 193: 265-275Abstract Full Text PDF PubMed Google Scholar). ACAT assays were also performed using a slightly modified protocol with cells maintained as stock cultures in medium B. For these experiments, the cells

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