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• W2079330956 abstract "This study has examined the dimeric/oligomeric forms of scavenger receptor class B type I (SR-BI) and its alternatively spliced form, SR-BII, in a diverse group of cells and tissues: i.e., normal and hormonally altered tissues of mice and rats as well as tissues of transgenic animals and genetically altered steroidogenic and nonsteroidogenic cells overexpressing the SR-B proteins. Using both biochemical and morphological techniques, we have seen that these dimeric and higher order oligomeric forms of SR-BI expression are strongly associated with both functional and morphological expression of the selective HDL cholesteryl ester uptake pathway. Rats and mice show some species differences in expression of SR-BII dimeric forms; this difference does not extend to the use of SR-B cDNA types for transfection purposes. In a separate study, cotransfection of HEK293 cells with cMyc and V5 epitope-tagged SR-BI permitted coprecipitation and quantitative coimmunocytochemical measurements at the electron microscope level, suggesting that much of the newly expressed SR-BI protein in stimulated cells dimerizes and that the SR-BI dimers are localized to the cell surface and specifically to microvillar or double membraned intracellular channels.These combined data suggest that SR-BI self-association represents an integral step in the selective cholesteryl ester uptake process. This study has examined the dimeric/oligomeric forms of scavenger receptor class B type I (SR-BI) and its alternatively spliced form, SR-BII, in a diverse group of cells and tissues: i.e., normal and hormonally altered tissues of mice and rats as well as tissues of transgenic animals and genetically altered steroidogenic and nonsteroidogenic cells overexpressing the SR-B proteins. Using both biochemical and morphological techniques, we have seen that these dimeric and higher order oligomeric forms of SR-BI expression are strongly associated with both functional and morphological expression of the selective HDL cholesteryl ester uptake pathway. Rats and mice show some species differences in expression of SR-BII dimeric forms; this difference does not extend to the use of SR-B cDNA types for transfection purposes. In a separate study, cotransfection of HEK293 cells with cMyc and V5 epitope-tagged SR-BI permitted coprecipitation and quantitative coimmunocytochemical measurements at the electron microscope level, suggesting that much of the newly expressed SR-BI protein in stimulated cells dimerizes and that the SR-BI dimers are localized to the cell surface and specifically to microvillar or double membraned intracellular channels. These combined data suggest that SR-BI self-association represents an integral step in the selective cholesteryl ester uptake process. The “selective” uptake of cholesteryl esters (CEs) from lipoprotein particles, such as HDL, is a process by which the HDL core CE is taken into the cells without the parallel uptake and degradation of the HDL particle itself (1Glass G. Pittman R.C. Weinstein D.B. Steinberg D. Dissociation of tissue uptake of cholesteryl ester from that of apoprotein A-I of rat plasma high density lipoprotein: selective delivery of cholesteryl ester to liver, adrenal, and gonad.Proc. Natl. Acad. Sci. USA. 1983; 80: 5435-5439Crossref PubMed Scopus (423) Google Scholar, 2Reaven E. Chen Y-D. I. Spicher M. Azhar S. Morphological evidence that high density lipoproteins are not internalized by steroid-producing cells during in situ organ perfusion.J. Clin. Invest. 1984; 74: 1387-1397Crossref Scopus (94) Google Scholar, 3Murakami M. Horiuchi S. Takata K. Morino Y. Distinction in the mode of receptor-mediated endocytosis between high density lipoprotein and acetylated high density lipoprotein: evidence for high density lipoprotein receptor-mediated cholesterol transfer.J. Biochem. 1987; 101: 729-741Crossref PubMed Scopus (59) Google Scholar, 4Pittman R.C. Knecht T.P. Rosenbaum M.S. Taylor Jr., C.A. A nonendocytotic mechanism for the selective uptake of high density lipoprotein-associated cholesterol esters.J. Biol. Chem. 1987; 262: 2443-2450Abstract Full Text PDF PubMed Google Scholar, 5Gwynne G.T. Mahaffee D.D. Rat adrenal uptake and metabolism of high density lipoprotein cholesteryl ester.J. Biol. Chem. 1989; 264: 8141-8150Abstract Full Text PDF PubMed Google Scholar). The pathway is a high-capacity, physiologically regulated, bulk cholesterol delivery system (6Azhar S. Reaven E. Scavenger receptor class BI and selective cholesteryl ester uptake: partners in the regulation of steroidogenesis.Mol. Cell. Endocrinol. 2002; 195: 1-26Crossref PubMed Scopus (159) Google Scholar, 7Azhar S. Leers-Sucheta S. Reaven E. Cholesterol uptake in adrenal and gonadal tissues: the SR-BI and 'selective’ pathway connection.Front. Biosci. 2003; 8: 998-1029Crossref Scopus (80) Google Scholar). Steroidogenic tissues (adrenal gland and gonads) of a variety of animal species, including mouse, hamster, cattle, and human, use this pathway to internalize HDL cholesterol for the production of steroid hormones (6Azhar S. Reaven E. Scavenger receptor class BI and selective cholesteryl ester uptake: partners in the regulation of steroidogenesis.Mol. Cell. Endocrinol. 2002; 195: 1-26Crossref PubMed Scopus (159) Google Scholar, 7Azhar S. Leers-Sucheta S. Reaven E. Cholesterol uptake in adrenal and gonadal tissues: the SR-BI and 'selective’ pathway connection.Front. Biosci. 2003; 8: 998-1029Crossref Scopus (80) Google Scholar); the pathway is used by the liver to mediate the transfer of cholesterol into bile (6Azhar S. Reaven E. Scavenger receptor class BI and selective cholesteryl ester uptake: partners in the regulation of steroidogenesis.Mol. Cell. Endocrinol. 2002; 195: 1-26Crossref PubMed Scopus (159) Google Scholar, 7Azhar S. Leers-Sucheta S. Reaven E. Cholesterol uptake in adrenal and gonadal tissues: the SR-BI and 'selective’ pathway connection.Front. Biosci. 2003; 8: 998-1029Crossref Scopus (80) Google Scholar). It operates also in isolated hepatocytes, fibroblasts, adipocytes, and macrophages, although its function in these cell systems is less clear (6Azhar S. Reaven E. Scavenger receptor class BI and selective cholesteryl ester uptake: partners in the regulation of steroidogenesis.Mol. Cell. Endocrinol. 2002; 195: 1-26Crossref PubMed Scopus (159) Google Scholar, 7Azhar S. Leers-Sucheta S. Reaven E. Cholesterol uptake in adrenal and gonadal tissues: the SR-BI and 'selective’ pathway connection.Front. Biosci. 2003; 8: 998-1029Crossref Scopus (80) Google Scholar). Scavenger receptor class B type I (SR-BI) is a physiologically relevant receptor identified for the selective pathway (6Azhar S. Reaven E. Scavenger receptor class BI and selective cholesteryl ester uptake: partners in the regulation of steroidogenesis.Mol. Cell. Endocrinol. 2002; 195: 1-26Crossref PubMed Scopus (159) Google Scholar, 7Azhar S. Leers-Sucheta S. Reaven E. Cholesterol uptake in adrenal and gonadal tissues: the SR-BI and 'selective’ pathway connection.Front. Biosci. 2003; 8: 998-1029Crossref Scopus (80) Google Scholar, 8Acton S. Rigotti A. Landschulz K.T. Xu S. Hobbs H.H. Krieger M. Identification of scavenger receptor SR-BI as a high density lipoprotein receptor.Science. 1996; 271: 518-520Crossref PubMed Scopus (2011) Google Scholar, 9Krieger M. Charting the fate of the “good cholesterol”: identification and characterization of the high-density lipoprotein receptor SR-BI.Annu. Rev. Biochem. 1999; 68: 523-558Crossref PubMed Scopus (460) Google Scholar, 10Williams D.L. Connelly M.A. Temel R.E. Swarnakar S. Phillips M.C. de la Llera-Moya M. Rothblat G.H. Scavenger receptor BI and cholesterol trafficking.Curr. Opin. Lipidol. 1999; 10: 329-339Crossref PubMed Scopus (173) Google Scholar, 11Silver D.L. Tall A.R. The cellular biology of scavenger receptor class B type I.Curr. Opin. Lipidol. 2001; 12: 497-504Crossref PubMed Scopus (75) Google Scholar). SR-BI is a member of the class B scavenger receptor family, all members of which contain a large extracellular domain that is anchored to the plasma membrane on each side by transmembrane domains adjacent to short cytoplasmic N- and C-terminal domains (6Azhar S. Reaven E. Scavenger receptor class BI and selective cholesteryl ester uptake: partners in the regulation of steroidogenesis.Mol. Cell. Endocrinol. 2002; 195: 1-26Crossref PubMed Scopus (159) Google Scholar, 7Azhar S. Leers-Sucheta S. Reaven E. Cholesterol uptake in adrenal and gonadal tissues: the SR-BI and 'selective’ pathway connection.Front. Biosci. 2003; 8: 998-1029Crossref Scopus (80) Google Scholar, 9Krieger M. Charting the fate of the “good cholesterol”: identification and characterization of the high-density lipoprotein receptor SR-BI.Annu. Rev. Biochem. 1999; 68: 523-558Crossref PubMed Scopus (460) Google Scholar, 10Williams D.L. Connelly M.A. Temel R.E. Swarnakar S. Phillips M.C. de la Llera-Moya M. Rothblat G.H. Scavenger receptor BI and cholesterol trafficking.Curr. Opin. Lipidol. 1999; 10: 329-339Crossref PubMed Scopus (173) Google Scholar, 11Silver D.L. Tall A.R. The cellular biology of scavenger receptor class B type I.Curr. Opin. Lipidol. 2001; 12: 497-504Crossref PubMed Scopus (75) Google Scholar). An alternatively spliced form of SR-BI, termed SR-BII or SR-BI.2, in which 40 entirely different amino acid residues replace 42 of the 45 C-terminal amino acid residues in the C-terminal cytoplasmic domain of SR-BI, has been described in several tissues, including certain cell types in which SR-BI expression is observed (12Webb N.R. de Villiers W.J.S. Connell P.M. de Beer F.C. van der Westhuyzen D.R. Alternative forms of the scavenger receptor BI (SR-BI).J. Lipid Res. 1997; 38: 1490-1495Abstract Full Text PDF PubMed Google Scholar). The primary function of this protein is not yet known, although it shows significant selective CE uptake function (13Webb N.R. Connell P.M. Graf G.A. Smart E.J. de Villiers W.J.S. de Beer F.C. van der Westhuyzen D.R. SR-BII, an isoform of the scavenger receptor BI containing an alternate cytoplasmic tail, mediates lipid transfer between high density lipoproteins and cells.J. Biol. Chem. 1998; 273: 15241-15248Abstract Full Text Full Text PDF PubMed Scopus (192) Google Scholar). It is of interest that steroidogenic tissues, which express high levels of SR-BI in vivo, are endowed with an intricate microvillar system for the trapping of lipoproteins. This general region of steroidogenic cells is referred to as the microvillar compartment, and the specialized space created between adjacent microvilli are called microvillar channels (14Reaven E. Boyles J. Spicher M. Azhar S. Evidence for surface entrapment of cholesterol-rich lipoproteins in luteinized ovary.Arteriosclerosis. 1988; 8: 298-309Crossref PubMed Google Scholar, 15Reaven E. Spicher M. Azhar S. Microvillar channels: a unique plasma membrane compartment for concentrating lipoproteins on the surface of rat adrenal cortical cells.J. Lipid Res. 1989; 30: 1551-1560Abstract Full Text PDF PubMed Google Scholar). It is in the microvillar channels that the various lipoproteins are trapped before the selective uptake of lipoprotein CEs into cells (14Reaven E. Boyles J. Spicher M. Azhar S. Evidence for surface entrapment of cholesterol-rich lipoproteins in luteinized ovary.Arteriosclerosis. 1988; 8: 298-309Crossref PubMed Google Scholar, 15Reaven E. Spicher M. Azhar S. Microvillar channels: a unique plasma membrane compartment for concentrating lipoproteins on the surface of rat adrenal cortical cells.J. Lipid Res. 1989; 30: 1551-1560Abstract Full Text PDF PubMed Google Scholar, 16Reaven E. Shi X-Y. Azhar S. Interaction of lipoproteins with isolated ovary plasma membranes.J. Biol. Chem. 1990; 265: 19100-19111Abstract Full Text PDF PubMed Google Scholar). Electron microscopic immunocytochemical techniques reveal heavy labeling for SR-BI specifically in these regions (17Reaven E. Nomoto A. Leers-Sucheta S. Temel R. Williams D.L. Azhar S. 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, 18Reaven E. Zhan L. Nomoto A. Leers-Sucheta S. Azhar S. Expression and microvillar localization of scavenger receptor class B, type I (SR-BI) and selective cholesteryl ester uptake in Leydig cells from rat testis.J. Lipid Res. 2000; 41: 343-356Abstract Full Text Full Text PDF PubMed Google Scholar, 19Azhar S. Nomoto A. Reaven E. Hormonal regulation of adrenal microvillar channel formation.J. Lipid Res. 2002; 43: 861-871Abstract Full Text Full Text PDF PubMed Google Scholar), and at present, there is no doubt that tissues with microvillar compartments expressing high levels of SR-BI are also active in selective CE uptake. Indeed, a recent study from this laboratory has shown that the relationship between microvillar channel formation, SR-BI content, and lipoprotein CE uptake in adrenal tissue of rats is hormone-regulated (19Azhar S. Nomoto A. Reaven E. Hormonal regulation of adrenal microvillar channel formation.J. Lipid Res. 2002; 43: 861-871Abstract Full Text Full Text PDF PubMed Google Scholar). Using hormonal stimulation and withdrawal regimens, we were able to manipulate adrenal SR-BI levels and carry out qualitative and quantitative measurements correlating SR-BI expression with microvillar mass and microvillar channel formation. Young male rats were used as controls, or hormone-stimulated with adrenocorticotropin (ACTH) for 24 h or with 17α-ethinyl estradiol for 5 days, or subjected to a withdrawal of ACTH hormones by dexamethasone treatment (24 h). Quantitative Western blot and immunocytochemistry analyses of adrenals from these animals indicated that ACTH and estradiol treatment greatly increased SR-BI expression (localized especially in the microvillar compartment of adrenocortical cells), whereas dexamethasone treatment led to decreased SR-BI. At the same time, striking ultrastructural changes occurred in the adrenocortical cell microvillar compartment, e.g., microvillar area, microvillar channel length, and microvillar complexity dramatically increased (compared with control values) after ACTH or estradiol treatment, whereas the same measurements showed major declines after dexamethasone treatment. It is of interest that adrenocortical cells of SR-BI knockout mice show microvillar changes similar to those seen in dexamethasone-treated rats (20Williams D.L. Wong J.S. Hamilton R.L. SR-BI is required for microvillar channel formation and the localization of HDL particles to the surface of adrenocortical cells in vivo.J. Lipid Res. 2002; 43: 544-549Abstract Full Text Full Text PDF PubMed Google Scholar). Western blots from these same adrenals also showed a striking difference in the expression of dimeric/oligomeric forms of SR-BI depending on hormonal treatment. (For simplicity, all dimeric and higher order oligomeric forms of SR-BI or SR-BII will be referred to as dimers in this report.) That is, samples of adrenal from animals treated with the stimulatory hormones showed from 2- to 10-fold higher levels of expression of SR-BI dimers/oligomers compared with controls, whereas samples from animals treated with dexamethasone showed far less SR-BI monomer or dimer forms than controls (19Azhar S. Nomoto A. Reaven E. Hormonal regulation of adrenal microvillar channel formation.J. Lipid Res. 2002; 43: 861-871Abstract Full Text Full Text PDF PubMed Google Scholar). Although Western blot analysis showing SR-BI dimers in adrenal had been observed previously by Landschulz et al. (21Landschulz K.T. Pathak R.K. Rigotti A. Krieger M. Hobbs H.H. Regulation of scavenger receptor, class B, type I, a high density lipoprotein receptor, in liver and steroidogenic tissues of the rat.J. Clin. Invest. 1996; 98: 984-995Crossref PubMed Scopus (470) Google Scholar), Williams et al. (22Williams D.L. de la Llera-Moya M. Thuahnai S.T. Lund-Katz S. Connelly M.A. Azhar S. Anantharamaiah G.M. Phillips M.C. Binding and cross-linking studies show that scavenger receptor BI interacts with multiple sites in apolipoprotein A-I and identify the class A amphipathic α-helix as a recognition motif.J. Biol. Chem. 2000; 275: 18897-18904Abstract Full Text Full Text PDF PubMed Scopus (100) Google Scholar), and our group after the use of one or another of these hormonal agents (19Azhar S. Nomoto A. Reaven E. Hormonal regulation of adrenal microvillar channel formation.J. Lipid Res. 2002; 43: 861-871Abstract Full Text Full Text PDF PubMed Google Scholar), the direct link between SR-BI dimer formation and architectural changes of the microvillar compartment, particularly changes in the extent of channel development, was both surprising and intriguing. Therefore, in the current study, we sought to characterize the monomeric/dimeric nature of the SR-BI complex in various tissues. Using both biochemical and morphological techniques, we have become convinced that dimeric forms of SR-BI expression are associated with various tissues active in selective CE uptake, and we describe here the dimeric expression of SR-BI (and in some cases its isoform SR-BII) in a diverse group of cells and tissues from normal and hormonally altered tissues of mice and rats as well as in tissues of transgenic animals and transfected cells overexpressing SR-BI. pcDNA6/myc-His, pcDNA6/V5-His, monoclonal anti-V5, and various cell culture media were obtained from Invitrogen Life Technologies (Carlsbad, CA). FUGENE 6 transfection reagent was purchased from Roche Diagnostics Corporation (Indianapolis, IN). The following reagents were supplied by Sigma Chemical Co. (St. Louis, MO): cholesteryl oleate, bovine plasma fibronectin, insulin, transferrin, human chorionic gonadotropin (hCG), Bt2cAMP, goat anti-rabbit IgG coupled to HRP, anti-cMyc, and galactose oxidase. The LumiGLO Chemiluminescent Substrate System used in Western blotting was obtained from KPL (Gaithersburg, MD). Rabbit anti-mouse IgG coupled to HRP was the product of Zymed (South San Francisco, CA). Goat anti-mouse IgG coupled to 10 nm colloidal gold and goat anti-rabbit IgG coupled to 15 nm colloidal gold were supplied by Ted Pella, Inc. (Reading, CA). All other reagents used were of analytical grade. The Sprague-Dawley male and female rats used in these studies were obtained from Harlan Sprague-Dawley (Indianapolis, IN). Mature (3 month old) rats were treated with a long-acting ACTH gel preparation (10 IU) at 0, 6, 12, and 24 h, or with vehicle alone, and the animals were killed 2 h after the last injection (19Azhar S. Nomoto A. Reaven E. Hormonal regulation of adrenal microvillar channel formation.J. Lipid Res. 2002; 43: 861-871Abstract Full Text Full Text PDF PubMed Google Scholar). The adrenals were removed, cleaned of adhering fat, and stored at −70°C until analyzed for SR-BI/SR-BII expression. Groups of 3 month old rats (or mice) were treated respectively with 25 IU (or 5 IU) of hCG (or with saline only) every 24 h for 4 days (18Reaven E. Zhan L. Nomoto A. Leers-Sucheta S. Azhar S. Expression and microvillar localization of scavenger receptor class B, type I (SR-BI) and selective cholesteryl ester uptake in Leydig cells from rat testis.J. Lipid Res. 2000; 41: 343-356Abstract Full Text Full Text PDF PubMed Google Scholar). These animals were killed on day 5, and their testes were excised, frozen, and stored at −70°C until analyzed for SR-BI and SR-BII protein expression by Western blotting. For ovary tissue, immature, 22–24 day old rats were injected subcutaneously with 50 IU of pregnant mare's serum gonadotropins (PMSG), followed 56 h later with 25 IU of hCG. Day 0 was considered the day of hCG injection. Such procedures result in superovulated (luteinized) ovaries by day 6 or 7 (17Reaven E. Nomoto A. Leers-Sucheta S. Temel R. Williams D.L. Azhar S. 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). For desensitized ovaries, the animals were injected with a second dose of hCG (25 IU) between 9:00 and 10:00 AM on day 6 (post-hCG), and ovaries were removed 24 h later (day 7) (17Reaven E. Nomoto A. Leers-Sucheta S. Temel R. Williams D.L. Azhar S. 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). Control animals received vehicle (saline) only. The freshly isolated ovaries were immediately homogenized and processed for the isolation of “light” and “heavy” membrane fractions using a discontinuous sucrose density gradient technique. For morphological studies, freshly excised adrenal, testes, and luteinized or desensitized ovarian samples were fixed and processed as described previously from this laboratory (17Reaven E. Nomoto A. Leers-Sucheta S. Temel R. Williams D.L. Azhar S. 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, 18Reaven E. Zhan L. Nomoto A. Leers-Sucheta S. Azhar S. Expression and microvillar localization of scavenger receptor class B, type I (SR-BI) and selective cholesteryl ester uptake in Leydig cells from rat testis.J. Lipid Res. 2000; 41: 343-356Abstract Full Text Full Text PDF PubMed Google Scholar, 19Azhar S. Nomoto A. Reaven E. Hormonal regulation of adrenal microvillar channel formation.J. Lipid Res. 2002; 43: 861-871Abstract Full Text Full Text PDF PubMed Google Scholar). The SR-BI transgenic mice (SR-BITg) used in this study were created in an FVB background and expressed the transgene specifically in the liver at levels ∼10-fold higher than those of control FVB mice (23Ueda Y. Royer L. Gong E. Zhang J. Cooper P.N. Francone O. Rubin E.M. Lower plasma levels and accelerated clearance of high density lipoprotein (HDL) and non-HDL cholesterol in scavenger receptor class B type I transgenic mice.J. Biol. Chem. 1999; 274: 7165-7171Abstract Full Text Full Text PDF PubMed Scopus (216) Google Scholar). These mice were kindly provided by Dr. Edward Rubin (Lawrence Berkeley National Laboratory, Berkeley, CA). For other studies, 3 month old male FVB mice were purchased from the Charles River Laboratories (Bloomington, IN). Immature female rats (21–23 days old) were injected subcutaneously with 17β-estradiol (1 mg) daily for 5 days (24Azhar 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 (62) Google Scholar, 25Reaven E. Tsai L. Azhar S. Intracellular events in the “selective” transport of lipoprotein-derived cholesteryl esters.J. Biol. Chem. 1996; 271: 16208-16217Abstract Full Text Full Text PDF PubMed Scopus (106) Google Scholar). The animals were killed 24 h after their last injection, and granulosa cells were isolated from ovaries and cultured for 72 h in Dulbecco's modified Eagle's medium: F12 medium supplemented with BSA (1 mg/ml), insulin (2 μg/ml), transferrin (5 μg/ml), hydrocortisone (100 ng/ml), and human fibronectin (2 μg/cm2), as described previously (24Azhar 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 (62) Google Scholar, 25Reaven E. Tsai L. Azhar S. Intracellular events in the “selective” transport of lipoprotein-derived cholesteryl esters.J. Biol. Chem. 1996; 271: 16208-16217Abstract Full Text Full Text PDF PubMed Scopus (106) Google Scholar). Subsequently, cells were treated with or without Bt2cAMP (2.5 mM) for an additional 24 h before the addition of other test substances. COS-7, CHO, and MLTC-1 cells were cultured in DMEM-10% fetal bovine serum, DMEM-F12-10% fetal bovine serum, and RPMI-1640-10% fetal bovine serum, respectively. R2C and Y1-BS1 cells were cultured in Ham's F10-15% equine serum and 2.5% fetal bovine serum. All media contained penicillin and streptomycin (1%). PCR primers against rat SR-BI nucleotide sequence (GenBank accession number D89655) were designed to facilitate the subcloning of full-length rat SR-BI cDNA into the pcDNA6/c-Myc-His and pcDNA6/V5-His version B vectors (Invitrogen). The forward primer, 5′-AAGCTTGCGCACGCGAACATGGGC-3′, contained a HindIII restriction site and was modified to generate a perfect Kozak sequence for efficient initiation of translation. The reverse primer, 5′-TCTAGACCCAGCTTGGCTTCTTGCAGTAC-3′, contained an XbaI restriction site, and the stop codon was mutated to encode a glycine residue to permit the production of SR-BI-c-myc or -V5 fusion protein. High-fidelity PCR was conducted using KOD HiFi DNA polymerase (Novagen) and rat SR-BI cDNA (26Reaven E. Leers-Sucheta S. Nomoto A. Azhar S. Expression of scavenger receptor class B, type I (SR-BI) promotes microvillar channel formation and selective cholesteryl ester transport in a heterologous reconstitution system.Proc. Natl. Acad. Sci. USA. 2001; 98: 1613-1618Crossref PubMed Scopus (61) Google Scholar) as template (6 ng). Two-step PCR cycling consisted of 25 cycles of denaturing and annealing (98°C for 20 s followed by 65°C for 30 s) without extension. The resulting cDNA was subcloned into the pSTBlue-1 vector (Novagen), sequenced on both strands, and then subcloned into the HindIII/XbaI site of the pcDNA6/c-myc-His and pcDNA6/V5-His vectors. The expression of SR-BI and SR-BII proteins in adrenal, liver, testes, ovary tissues, and cultured cells was assessed by Western blot analysis using previously described methodology (17Reaven E. Nomoto A. Leers-Sucheta S. Temel R. Williams D.L. Azhar S. 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, 18Reaven E. Zhan L. Nomoto A. Leers-Sucheta S. Azhar S. Expression and microvillar localization of scavenger receptor class B, type I (SR-BI) and selective cholesteryl ester uptake in Leydig cells from rat testis.J. Lipid Res. 2000; 41: 343-356Abstract Full Text Full Text PDF PubMed Google Scholar, 19Azhar S. Nomoto A. Reaven E. Hormonal regulation of adrenal microvillar channel formation.J. Lipid Res. 2002; 43: 861-871Abstract Full Text Full Text PDF PubMed Google Scholar, 27Azhar S. Nomoto A. Leers-Sucheta S. Reaven E. Simultaneous induction of an HDL receptor protein (SR-BI) and the selective uptake of HDL-cholesteryl esters in a physiologically relevant steroidogenic cell model.J. Lipid Res. 1998; 39: 1616-1628Abstract Full Text Full Text PDF PubMed Google Scholar). Briefly, intact tissues were homogenized in 10 volumes of buffer (20 mM Tris-HCl, pH 7.5, 2 mM MgCl2, 0.25 M sucrose, 1 mM PMSF, 10 μg/ml leupeptin, 20 μg/ml aprotinin, and 5 μg/ml pepstatin) and centrifuged (800 g) for 10 min, and the supernatant was centrifuged for 60 min at 100,000 g. The resulting pellet was washed and resuspended in buffer, and the resulting membrane samples were used for immunoblotting of SR-BI and SR-BII. Similarly, cultured cells were washed twice in ice-cold phosphate-buffered saline and lysed in lysis buffer (125 mM Tris-HCl, pH 6.8, 2% SDS, 5% glycerol, 1% 2-mercaptoethanol, 100 mM PMSF, 10 μg/ml leupeptin, 20 μg/ml aprotinin, and 5 μg/ml pepstatin A). After incubation at 37°C for 15 min, each lysate was sonicated briefly to disrupt chromatin (DNA) and then used for immunoblotting. Aliquots of tissue, cell, or isolated membrane samples were mixed with equal volumes of 2× Laemmli sample buffer [20 mM Tris-HCl, pH 6.8, 2% SDS (w/v), 10% sucrose (w/v), and 1% 2-mercaptoethanol] and subjected to 7% SDS-PAGE. For each sample, a constant amount of protein (2–40 μg) was loaded on the gel. Protein standards (myosin, 200 kDa; β-galactosidase, 116.3 kDa; phosphorylase b, 97.4 kDa; BSA, 66.2 kDa; and ovalbumin, 45 kDa) were also loaded on the gel. After electrophoretic separation, the proteins were transferred to Immobilon polyvinylidene difluoride (PVDF) membranes using standard techniques. The protein blots were incubated with anti-SR-BI or anti-SR-BII for 2 h at room temperature, probed with peroxidase-labeled mouse anti-rabbit IgG, and visualized using the enhanced chemiluminescence (ECL) system. The resulting radiographic chemiluminescence was visualized for different time points (1–10 min), and appropriate films were subjected to densitometric scanning. Blue native-PAGE (BN-PAGE) was carried out using a slight modification (28Yang T. Espenshade P.J. Wright M.E. Yabe D. Gong Y. Aebersold R. Goldstein J.L. Brown M.S. Crucial step in cholesterol homeostasis: sterols promote binding of SCAP to INSIG-1, a membrane protein that facilitates retention of SREBPs in ER.Cell. 2002; 110: 489-500Abstract Full Text Full Text PDF PubMed Scopus (795) Google Scholar) of the procedure of Schägger, Cramer, and von Jagow (29Schägger H. Cramer W.A. von Jagow G. Analysis of molecular masses and oligomeric states of protein complexes by blue native electrophoresis and isolation of membrane protein complexes by two-dimensional native electrophoresis.Anal. Biochem. 1994; 217: 220-230Crossref PubMed Scopus (1043) Google Scholar). In brief, aliquots of 100 μg protein samples in 100 μl volume were mixed with 100 μl of buffer containing 40 mM HEPES-KOH, pH 7.0, 2 mM magnesium acetate, and 2% (w/v) digitonin in a final volume of 200 μl. After incubation at 4°C for 30 min, detergent-insoluble material was removed by centrifugation at 20,000 g for 20 min at 4°C. One aliquot of the" @default.
• W2079330956 created "2016-06-24" @default.
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• W2079330956 creator A5039155716 @default.
• W2079330956 creator A5049078893 @default.
• W2079330956 creator A5077425218 @default.
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• W2079330956 date "2004-03-01" @default.
• W2079330956 modified "2023-10-16" @default.
• W2079330956 title "Dimerization of the scavenger receptor class B type I" @default.
• W2079330956 cites W1492059346 @default.
• W2079330956 cites W1523684025 @default.
• W2079330956 cites W1534257883 @default.
• W2079330956 cites W1571586310 @default.
• W2079330956 cites W1576930851 @default.
• W2079330956 cites W1656852118 @default.
• W2079330956 cites W1824441884 @default.
• W2079330956 cites W1870049952 @default.
• W2079330956 cites W1963530308 @default.
• W2079330956 cites W1968883066 @default.
• W2079330956 cites W1974711948 @default.
• W2079330956 cites W1983125054 @default.
• W2079330956 cites W1985170971 @default.
• W2079330956 cites W1987920070 @default.
• W2079330956 cites W1999307352 @default.
• W2079330956 cites W2001995220 @default.
• W2079330956 cites W2004805723 @default.
• W2079330956 cites W2016597009 @default.
• W2079330956 cites W2019560803 @default.
• W2079330956 cites W2021133118 @default.
• W2079330956 cites W2031423487 @default.
• W2079330956 cites W2034999042 @default.
• W2079330956 cites W2039486449 @default.
• W2079330956 cites W2043476739 @default.
• W2079330956 cites W2045681007 @default.
• W2079330956 cites W2048163544 @default.
• W2079330956 cites W2050284220 @default.
• W2079330956 cites W2052551760 @default.
• W2079330956 cites W2054407280 @default.
• W2079330956 cites W2062742229 @default.
• W2079330956 cites W2063358283 @default.
• W2079330956 cites W2076959859 @default.
• W2079330956 cites W2076976482 @default.
• W2079330956 cites W2077817909 @default.
• W2079330956 cites W2083446323 @default.
• W2079330956 cites W2091958625 @default.
• W2079330956 cites W2097097666 @default.
• W2079330956 cites W2097176143 @default.
• W2079330956 cites W2097632838 @default.
• W2079330956 cites W2100837269 @default.
• W2079330956 cites W2102131116 @default.
• W2079330956 cites W2102557954 @default.
• W2079330956 cites W2121080720 @default.
• W2079330956 cites W2125856024 @default.
• W2079330956 cites W2132116708 @default.
• W2079330956 cites W2133744342 @default.
• W2079330956 cites W2156818917 @default.
• W2079330956 cites W2158207659 @default.
• W2079330956 cites W2163369915 @default.
• W2079330956 cites W2166004416 @default.
• W2079330956 cites W2186760046 @default.
• W2079330956 cites W2306336891 @default.
• W2079330956 cites W2324147954 @default.
• W2079330956 cites W2325770196 @default.
• W2079330956 cites W2418418368 @default.
• W2079330956 cites W2468239922 @default.
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