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• W2024692118 abstract "Previously we reported that when cell cholesterol is acutely lowered with β-methyl-cyclodextrin the amount of activated ERK1/2 in caveolae dramatically increases. We traced the origin of this novel method of pERK1/2 accumulation to a macromolecular complex with dual specific phosphatase activity that contains the serine/threonine phosphatase PP2A, the tyrosine phosphatase HePTP, the oxysterol-binding protein OSBP and cholesterol. When cell cholesterol is lowered, or oxysterols is introduced, the complex disassembles and pERK1/2 increases. In an effort to better understand how OSBP functions as a cholesterol-regulated scaffolding protein, we have mapped the functional parts of the molecule. The command center of the molecule is a centrally located, 51 amino acids (408-459) long sterol-binding domain that can bind both cholesterol and 25-hydroxycholesterol. This domain is functional whether attached to the N- or the C-terminal half of OSBP. Introduction of a Y458S mutation impairs binding. Even though 25-hydroxycholesterol will compete for cholesterol binding to OSBP408-809, it will not compete for cholesterol binding in full-length OSBP. Upon further analysis we found that a glycine-alaninerich region at the N-terminal end of OSBP works with the PH domain to control cholesterol binding without affecting 25-hydroxycholesterol binding. Finally, we found that HePTP and PP2A bind the C-terminal half of OSBP, HePTP binds a coiled-coil domain (amino acids 732-761), and PP2A binds neither the coiled-coil nor HePTP. On the basis of this information we propose a new model for how OSBP is able to sense both membrane cholesterol and oxidized sterols and link this information to the ERK1/2 signaling pathway. Previously we reported that when cell cholesterol is acutely lowered with β-methyl-cyclodextrin the amount of activated ERK1/2 in caveolae dramatically increases. We traced the origin of this novel method of pERK1/2 accumulation to a macromolecular complex with dual specific phosphatase activity that contains the serine/threonine phosphatase PP2A, the tyrosine phosphatase HePTP, the oxysterol-binding protein OSBP and cholesterol. When cell cholesterol is lowered, or oxysterols is introduced, the complex disassembles and pERK1/2 increases. In an effort to better understand how OSBP functions as a cholesterol-regulated scaffolding protein, we have mapped the functional parts of the molecule. The command center of the molecule is a centrally located, 51 amino acids (408-459) long sterol-binding domain that can bind both cholesterol and 25-hydroxycholesterol. This domain is functional whether attached to the N- or the C-terminal half of OSBP. Introduction of a Y458S mutation impairs binding. Even though 25-hydroxycholesterol will compete for cholesterol binding to OSBP408-809, it will not compete for cholesterol binding in full-length OSBP. Upon further analysis we found that a glycine-alaninerich region at the N-terminal end of OSBP works with the PH domain to control cholesterol binding without affecting 25-hydroxycholesterol binding. Finally, we found that HePTP and PP2A bind the C-terminal half of OSBP, HePTP binds a coiled-coil domain (amino acids 732-761), and PP2A binds neither the coiled-coil nor HePTP. On the basis of this information we propose a new model for how OSBP is able to sense both membrane cholesterol and oxidized sterols and link this information to the ERK1/2 signaling pathway. Recently we proposed a model for how sterol binding to oxysterol-binding protein (OSBP) 2The abbreviations used are: OSBPoxysterol-binding proteinERKextracellular signal-regulated kinaseERendoplasmic reticulumORPOSBP-related proteinHAhemagglutininTricineN-[2-hydroxy-1,1-bis(hydroxymethyl)ethyl]glycineNTAnitrilotriacetic acidANOVAanalysis of variance. is linked to a critical signaling pathway in the cell (1Wang P.Y. Weng J. Anderson R.G. Science. 2005; 307: 1472-1476Crossref PubMed Scopus (247) Google Scholar). Conditions that lower the cholesterol level of the plasma membrane (in particular, caveolae cholesterol) cause a dramatic increase in the level of cellular pERK1/2, which is followed by a marked increase in DNA synthesis (2Furuchi T. Anderson R.G. J. Biol. Chem. 1998; 273: 21099-21104Abstract Full Text Full Text PDF PubMed Scopus (336) Google Scholar). The cause of the pERK1/2 increase is the loss of an enzyme activity that is able to remove phosphate from both threonine 185 and tyrosine 187 in human pERK2 (3Wang P.Y. Liu P. Weng J. Sontag E. Anderson R.G. EMBO J. 2003; 22: 2658-2667Crossref PubMed Scopus (69) Google Scholar). Instead of being a single protein, however, the dual phosphatase activity involves two separate sets of enzymes. One is the multiprotein, threonine phosphatase PP2A. The other is a member of the PTPPBS family (4Augustine K.A. Silbiger S.M. Bucay N. Ulias L. Boynton A. Trebasky L.D. Medlock E.S. Anat. Rec. 2000; 258: 221-234Crossref PubMed Scopus (23) Google Scholar) of tyrosine phosphatase. These proteins work cooperatively as part of a high molecular weight complex (∼440 kDa) to dephosphorylate pERK1/2 when plasma membrane cholesterol is normal. Cholesterol depletion, however, silences the dual specific activity and, concomitant with the loss of cholesterol, the high molecular weight complex disassembles. Therefore, the cooperative behavior of the two enzymes is dependent on a cholesterol binding, scaffolding protein that holds the two phosphatases in the active configuration only when cholesterol is bound. A protein with the expected scaffolding activity was identified as OSBP (1Wang P.Y. Weng J. Anderson R.G. Science. 2005; 307: 1472-1476Crossref PubMed Scopus (247) Google Scholar). oxysterol-binding protein extracellular signal-regulated kinase endoplasmic reticulum OSBP-related protein hemagglutinin N-[2-hydroxy-1,1-bis(hydroxymethyl)ethyl]glycine nitrilotriacetic acid analysis of variance. A lipid-sensing protein is one where the occupation of a binding pocket by a specific lipid causes the molecule to undergo a conformational change that conveys new functional activity. A well characterized example of this type of molecule is the sterol-sensor family of membrane proteins (5Goldstein J.L. DeBose-Boyd R.A. Brown M.S. Cell. 2006; 124: 35-46Abstract Full Text Full Text PDF PubMed Scopus (1246) Google Scholar). These are polytopic membrane proteins that contain a sterol-sensing motif within the spanning regions. In the case of the sterol sensors SCAP and HMG-CoA reductase, cholesterol binding to this motif causes each to bind one of the two isoforms of the integral membrane protein Insig. Whereas the binding of Insig to HMG-CoA reductase stimulates reductase degradation, Insig binding to SCAP causes retention of the transcription factor SREBP in the ER. Importantly, these sterol sensors appear to be specialized to sense the local level of sterol in the membrane where they reside (5Goldstein J.L. DeBose-Boyd R.A. Brown M.S. Cell. 2006; 124: 35-46Abstract Full Text Full Text PDF PubMed Scopus (1246) Google Scholar). In contrast to SCAP and HMG-CoA reductase, our work (1Wang P.Y. Weng J. Anderson R.G. Science. 2005; 307: 1472-1476Crossref PubMed Scopus (247) Google Scholar) and others (6Mohammadi A. Perry R.J. Storey M.K. Cook H.W. Byers D.M. Ridgway N.D. J. Lipid Res. 2001; 42: 1062-1071Abstract Full Text Full Text PDF PubMed Google Scholar, 7Perry R.J. Ridgway N.D. Mol. Biol. Cell. 2006; 17: 2604-2616Crossref PubMed Scopus (198) Google Scholar, 8Yan D. Lehto M. Rasilainen L. Metso J. Ehnholm C. Yla-Herttuala S. Jauhiainen M. Olkkonen V.M. Arterioscler. Thromb. Vasc. Biol. 2007; 27: 1108-1114Crossref PubMed Scopus (64) Google Scholar) suggests OSBP is a cytosolic sterol sensor. OSBP is a member of a protein family (referred to as OSBP-related proteins (ORPs)) that is widespread in nature with sixteen in humans, six in plants, and seven in yeast (9Levine T. Mol. Cell. 2005; 19: 722-723Abstract Full Text Full Text PDF PubMed Scopus (6) Google Scholar). OSBP is 809 amino acids long (Fig. 1) and contains an N terminus (1-88) rich in glycine and alanine (Gly-Ala domain), a PH domain (90-183), a leucine zipper (209-244), a FFAT motif (360-364) that binds the ER protein VAP, and a sterol-binding region (408-809). Originally discovered as an oxysterol-binding protein (10Dawson P.A. Van der Westhuyzen D.R. Goldstein J.L. Brown M.S. J. Biol. Chem. 1989; 264: 9046-9052Abstract Full Text PDF PubMed Google Scholar, 11Taylor F.R. Kandutsch A.A. Chem. Phys. Lipids. 1985; 38: 187-194Crossref PubMed Scopus (76) Google Scholar), the crystal structure of the yeast ORP Osh4 indicates that the sterol-binding pocket of OSBP may be able to accommodate a variety of sterols including cholesterol. The principle function of OSBP and other ORPs is thought to be to move sterols between different membrane compartments, although experiments in yeast “suggest that elimination of Osh proteins has an impact on the ability of the PM to sequester sterols but does not directly affect the transport of sterols” (12Sullivan D.P. Ohvo-Rekila H. Baumann N.A. Beh C.T. Menon A.K. Biochem. Soc. Trans. 2006; 34: 356-358Crossref PubMed Scopus (74) Google Scholar). Here we present new evidence in support of our model that OSBP functions as a sterol sensor. We map the sterol-binding region of OSBP to an area between residue 409 and 459 and show that it is this sequence that conveys stereospecific cholesterol and oxysterol binding. The binding site for the two OSBP client enzymes (HePTP and PP2A) resides between 459 and 809. Finally, we present evidence that the alanine/glycine-rich region at the N terminus (10Dawson P.A. Van der Westhuyzen D.R. Goldstein J.L. Brown M.S. J. Biol. Chem. 1989; 264: 9046-9052Abstract Full Text PDF PubMed Google Scholar) controls the binding of cholesterol, but not 25-hydroxycholesterol, to the sterol-binding site in OSBP. Materials—Monoclonal anti-V5 antibody was from Invitrogen (Carlsbad, CA). Monoclonal Abs for HA and Myc were from UBI (Lake Placid, NY). PP2A, C mAb was from BD Biosciences (San Jose, CA) and PP2A, B55α, was a gift from Dr. Estelle Sontag at the University of Texas Southwestern Medical Center. Poly-Prep chromatography columns were from Bio-Rad and Ni-NTA beads were purchased from Qiagen (Valencia, CA). Cholesterol and 25-hydroxycholesterol were from Sigma. Epicholesterol was from Steraloids Inc (Newport, RI). 1, 2-[3H]-(N)-cholesterol and 25-(26, 27-[3H])hydroxycholesterol were from PerkinElmer (Boston, MA). Protease inhibitor mixture set III and PMSF were from Calbiochem. Construction of Recombinant OSBPs—The key OSBP cDNA constructs used in this study, which were derived from a rabbit sequence (13Dawson P.A. Ridgway N.D. Slaughter C.A. Brown M.S. Goldstein J.L. J. Biol. Chem. 1989; 264: 16798-16803Abstract Full Text PDF PubMed Google Scholar), are shown schematically in supplemental Fig. S3. For bacterial-expressed protein, all clones were inserted into the pET102-V5-His bacterial expression vector and expressed in Escherichia coli strain BL21 or Rosetta. pET102-OSBP-V5-His was constructed as previously described (1Wang P.Y. Weng J. Anderson R.G. Science. 2005; 307: 1472-1476Crossref PubMed Scopus (247) Google Scholar). pET102-OSBP-V5-His was used as the template to generate a set of subclones. To obtain pET102-OSBP1-473-V5-His, the pET102-OSBP-V5-His was digested with HindIII, partially digested with XbaI, and the 5′ overhang of the purified large XbaI-HindIII fragment was filled in and ligated. To obtain pET102-OSBP1Δ77-310-V5-His, the pET102-OSBP-V5-His was digested with XhoI, partially digested with SgrAI, and the 5′ overhangs of the purified large SgrAI-XhoI fragment was filled in and ligated. To obtain the pET102-OSBP459-809-V5-His, pET102-OSBP-V5-His was digested with NcoI and the 5′ overhangs of the purified larger NcoI fragment was filled in and ligated. PCR was performed to obtain pET102-OSBP1-409-V5-His, pET102-OSBP311-809-V5-His, pET102-OSBP408-809-V5-His, pET102-OSBPY298S-V5-His, pET102-OSBPY458S-V5-His, and pET102-OSBP181-809. The clones were constructed using a QuikChange Site-Directed Mutagenesis kit (Stratagene, La Jolla, CA) with the following PCR primers respectively; 5′-CCAAGAAGGAAAAGAGAACCGGTAAGCCTATCCCTAACCC-3′ forward, 5′-GTTTAAGAAGGAGATATACATAATGGAGCAGCTGGCAAAGC-3′ forward, 5′-AAGAAGGAGATATACATAATGGGGAGAACCAGAATACCATACAAG-3′ forward, 5′-GCAGAAGTCGCTGCAGTCTGAGAGAGACCAGCGC-3′ forward, 5′-GCCTTACTGAAGATCTGGAATCCCATGGGCTGTTAGACCG-3′ forward and 5′-CTTTAAGAAGGAGATATACATAATGGCCAAGGCCAAGGCTGTG-3′ forward. pET102-OSBPΔ87-182-V5-His was constructed as previously described (1Wang P.Y. Weng J. Anderson R.G. Science. 2005; 307: 1472-1476Crossref PubMed Scopus (247) Google Scholar). For the co-expression studies in HeLa cells, we used the pcDNA3.1-myc-His mammalian expression vector. pcDNA3.1-OSBP408-809-myc-His and pcDNA3.1-OSBP1-473-myc-His were constructed as described above for the pET102 vectors. pcDNA3.1-OSBPΔ732-760 was constructed by PCR using the primer forward 5′-GGAAAATGGGCGCTGGGAAGATGGCACACCTTATGATCC-3′. All subcloned cDNAs were sequenced to verify that the coding sequence was correct. Sterol Binding Assay—Expression of cDNA inserts in pET102 is induced by isopropyl-1-thio-β-d-galactopyranoside for 3-4 h at room temperature. Bacteria were collected and resuspended in buffer A (50 mm NaH2PO4, 300 mm NaCl, pH 7.9, 10 mm imidazole, 0.2% protease inhibitor mixture set III, and 1 mm phenylmethylsulfonyl fluoride). Bacteria were disrupted by sonication and centrifuged at 4,000 × g for 25 min to remove debris. The supernatant fraction was further centrifuged at 200,000 × g for 30 min. The clear supernatant fraction was assayed for protein concentration and OSBP construct expression level before each experiment. For each binding assay, the protein concentration of the lysate was kept at 1.8 mg/ml. To achieve similar concentrations, the constructs expression level was checked by semi-quantitative immunoblotting, and the highest expressing construct in each experiment was diluted with bacterial lysates from non-expressing cells. Sterol binding was measured by placing 9.5 ml of lysate in a 15-ml conical tube, adding the indicated concentration of [3H]sterol in 20 μl of 100% ethanol, and rotating the sample for 2 h at room temperature. Subsequently, 200 μl of Ni-NTA beads (prepared according to the manufacturer's instruction) was added to each tube, and the rotation continued for an additional 1 h at 4 °C. The sample was then centrifuged at 500 × g for 2 min to sediment the beads, and the supernatant fraction designated the unbound fraction. The beads were resuspended in buffer A and loaded onto a poly-prep chromatography column. Following three washings with 10 mm imidazole, once with 20 mm imidazole and once with 40 mm imidazole in buffer A, the bound His (6Mohammadi A. Perry R.J. Storey M.K. Cook H.W. Byers D.M. Ridgway N.D. J. Lipid Res. 2001; 42: 1062-1071Abstract Full Text Full Text PDF PubMed Google Scholar)-tagged proteins were eluted with 160 mm imidazole in buffer A. The tritium radioactivity in the elution was then measured by scintillation counting. Instead of using the columns, the beads can also be washed by low speed centrifugation (500 × g, 2 min) using the same washing procedure. Then the beads were resuspended in 160 mm imidazole and centrifuged at 500 × g for 2 min. The radioactivity in the supernatant/elution was determined by scintillation counting. Immunoblotting with a V5 mAb was performed to confirm equal amounts of His6-tagged OSBP protein in each assay of the same experiment. For each experiment, the lysate from non-expressing/control cells was employed for a parallel binding assay and the radioactivity in the elution was considered as the background. OSBP-specific 3H binding was obtained by subtracting the background radioactivity from the total radioactivity associated with the OSBP samples. The OSBP-specific bound tritium was then converted into fmol of sterol. Each binding curve was plotted with GraphPad Prism software, and the binding Kd was estimated using a nonlinear regression curve. With the Bradford protein assay, we estimated that 10-20 μg of OSBP construct was present in each assay and that the final ethanol concentration in each experiment was ∼0.5%. In tests not shown, we found that as much as 2% ethanol had no effect on the sterol binding characteristics of both wild type and mutant OSBPs. Depending on the sterol affinity and expression level, background binding (bacterial lysate alone) values for 25-hydroxycholesterol ranged from 1-18% and for cholesterol from 4-26%. A compilation of the binding experiments for the various constructs is shown in supplemental Fig. S3. OSBP-HePTP/PP2A Interaction Assay—The HePTP/PP2A-OSBP interaction assay was carried out in HeLa cells as previously described. Briefly cells were transfected with the indicated cDNA using Superfect transfection reagent, cultured for 24 h, washed with phosphate-buffered saline, resuspended in Buffer B (20 mm Tricine, pH 7.8, 250 mm sucrose, 1 mm EDTA, and 0.5 mm phenylmethylsulfonyl fluoride), and homogenized by nitrogen bomb at 500 psi for 15 min on ice. The homogenates were centrifuged for 30 min at 200,000 × g and the His-tagged OSBP construct isolated from the supernatant fraction using Ni-NTA beads as previously described (1Wang P.Y. Weng J. Anderson R.G. Science. 2005; 307: 1472-1476Crossref PubMed Scopus (247) Google Scholar). Other Methods—We used a one-way ANOVA followed by multiple comparisons using Fisher's Least-Significant-Difference Test to analyze sterol binding to both normal and mutant OSBP (supplemental Fig. S1). Localization of the Sterol-binding Site—We reported previously that cholesterol promotes assembly of OSBP/HePTP/PP2A while 25-hydroxycholesterol causes disassembly (1Wang P.Y. Weng J. Anderson R.G. Science. 2005; 307: 1472-1476Crossref PubMed Scopus (247) Google Scholar), which implies that both sterols interact with OSBP. Recently the crystal structure of Osh4 was solved (14Im Y.J. Raychaudhuri S. Prinz W.A. Hurley J.H. Nature. 2005; 437: 154-158Crossref PubMed Scopus (340) Google Scholar) and a sterol-binding pocket defined (Fig. 2). Consistent with our observation, this ORP can bind both cholesterol and oxysterol. The region of OSBP that is homologous to Osh4 (Fig. 2) encompasses amino acids 408-809 (OSBP408-809) and contains the previously defined oxysterol-binding region of the molecule (13Dawson P.A. Ridgway N.D. Slaughter C.A. Brown M.S. Goldstein J.L. J. Biol. Chem. 1989; 264: 16798-16803Abstract Full Text PDF PubMed Google Scholar, 15Ridgway N.D. Dawson P.A. Ho Y.K. Brown M.S. Goldstein J.L. J. Cell Biol. 1992; 116: 307-319Crossref PubMed Scopus (241) Google Scholar, 16Suchanek M. Hynynen R. Wohlfahrt G. Lehto M. Johansson M. Saarinen H. Radzikowska A. Thiele C. Olkkonen V.M. Biochem. J. 2007; 405: 473-480Crossref PubMed Scopus (121) Google Scholar). To see if OSBP408-809 will also bind cholesterol, we developed a method for measuring sterol binding to bacterial expressed OSBP408-809 (see “Experimental Procedures”). We tested the method by measuring the ability of OSBP408-809 to bind 25-[3H]hydroxycholesterol as a function of sterol concentration (Fig. 3A). Binding was saturable with an average half-maximal binding (apparent Kd) of 5 nm (Fig. 3C), which is comparable to the binding of 25-hydroxycholesterol to full-length OSBP expressed in COS-M6 cells (13Dawson P.A. Ridgway N.D. Slaughter C.A. Brown M.S. Goldstein J.L. J. Biol. Chem. 1989; 264: 16798-16803Abstract Full Text PDF PubMed Google Scholar). OSBP408-809 also bound cholesterol (Fig. 3B). A competition binding assay was used to determine if cholesterol and 25-hydroxycholesterol bound to the same site on the molecule (Fig. 3D). Both unlabeled cholesterol and 25-hydroxycholesterol competed for [3H]cholesterol binding to OSBP408-809. Epicholesterol, which is an optical isomer of cholesterol, failed to compete for binding. On the other hand, cholesterol did not compete for binding of 25-hydroxycholesterol to OSBP (E). We do not know the reason for this anomaly, but it may be due to the low solubility and low affinity of cholesterol compared with 25-hydroxycholesterol. Nevertheless, OSBP408-809 appears to have a single, stereospecific, sterol-binding site that can bind either cholesterol or oxysterols.FIGURE 3Stereo-specific binding of cholesterol (B, C-E) and 25-hydroxycholesterol (A, C-E) to OSBP408-809. In all experiments we used lysate from either bacteria-expressing His-tagged OSBP408-809 or control bacteria as described. A, constant amount of each lysate was incubated in the presence of the indicated concentration of 25-OH-[3H]cholesterol for 2 h at room temperature, and purified by nickel chromatography. Specific binding was calculated by subtracting the [3H] associated with the control sample. B, same protocol was used to measure [3H]cholesterol binding. C, diagram of the OSBP408-809 construct along with its average estimated binding affinity of 25-hydroxycholesterol and cholesterol. D, OSBP408-809 was incubated with 30 nm [3H]cholesterol in the presence of the indicated concentrations of unlabeled cholesterol (•), 25-hydroxycholesterol (▵), or epicholesterol (▪). The OSBP408-809-specific bound [3H]cholesterol was determined by scintillation counting. E, OSBP408-809 was incubated with 5 nm 25-OH-[3H]cholesterol in the presence of the indicated concentrations of unlabeled cholesterol (^), 25-hydroxycholesterol (▵), or epicholesterol (▪) for 2 h at room temperature. The OSBP408-809-specific bound 25-OH-[3H]cholesterol was determined by scintillation counting. We used immunoblotting to confirm that each sample within an experiment had the same amount of expressed OSBP constructs.View Large Image Figure ViewerDownload Hi-res image Download (PPT) The ability of OSBP408-809 to bind both cholesterol and 25-hydroxycholesterol is in agreement with the structural properties of the sterol-binding pocket found in Osh4 (14Im Y.J. Raychaudhuri S. Prinz W.A. Hurley J.H. Nature. 2005; 437: 154-158Crossref PubMed Scopus (340) Google Scholar). Osh4 predicts that the β-barrel region of OSBP between amino acids 458 and 694 is the sterol-binding site (Fig. 2). A convenient restriction site allowed us to construct a cDNA coding for amino acids 1-473 of OSBP (OSBP1-473), which does not include any part of the the β-barrel region (Fig. 2). This construct clearly binds 25-hydroxycholesterol (Fig. 4A and supplemental Figs. S1 and S3) with an average Kd of 122 nm (Fig. 4C and supplemental Fig. S1). It also binds cholesterol (•, Fig. 4B) with a Kd of ∼216 nm. If, on the other hand, the first 458 amino acids of OSBP (OSBP459-809) is deleted, sterol binding is lost (▵, Fig. 4, B and C). We also did not detect sterol binding to amino acids 1-409 (Figs. 4C, 5B, and 6A). The lack of sterol binding to both OSBP1-409 and OSBP459-809 suggests there is a single sterol-binding site in OSBP between amino acid 408 and 459 that will bind sterol when attached to either the N-terminal or the C-terminal one-half of the molecule. According to the model of Osh4 (14Im Y.J. Raychaudhuri S. Prinz W.A. Hurley J.H. Nature. 2005; 437: 154-158Crossref PubMed Scopus (340) Google Scholar), OSBP409-459 maps to the putative lid region of OSBP (Fig. 2). Based on this analysis, the β barrel region is not the primary sterol-binding site in OSBP.FIGURE 5Full-length OSBP binds both cholesterol and 25-hydroxycholesterol. Experiments were carried out as described in the legend to Fig. 3. A, constant amount of each lysate was incubated in the presence of the indicated concentration of 25-OH-[3H]cholesterol for 2 h at room temperature, and purified by nickel chromatography. Specific binding was calculated by subtracting the [3H] associated with the control lysate. B, same protocol was used to measure [3H]cholesterol binding to the full-length OSBP. As a negative control, we measured [3H]cholesterol binding to OSBP1-409 (▪). C, diagram of full-length OSBP with the average estimated binding affinity of 25-hydroxycholesterol and cholesterol. A one-way ANOVA followed by multiple comparisons using Fisher's Least-Significant-Difference Test shows that the binding of sterol to 1-809 is statistically significant (supplemental Fig. S1). D, same sterol binding protocol was used to measure 25-OH-[3H]cholesterol binding to two mutated full-length OSBPs (Y298S and Y458S). Kd for binding of Y298S was 15.2 ± 1.2 nm. E, full-length OSBP was incubated in the presence of 60 nm [3H]cholesterol plus the indicated concentration of unlabeled 25-hydroxycholesterol for 2 h at room temperature. The amount of specific [3H]cholesterol bound was measured. We used immunoblotting to confirm that each sample within an experiment had the same amount of expressed OSBP constructs.View Large Image Figure ViewerDownload Hi-res image Download (PPT)FIGURE 6The N terminus of OSBP regulates cholesterol binding. A, same protocol as described above was used to measure 25-OH-[3H]cholesterol binding to OSBP311-809 (▪) and OSBP1-409 (▴). B, OSBP311-809 was incubated in the presence of 30 nm [3H]cholesterol plus the indicated concentration of unlabeled cholesterol (•), 25-hydroxycholesterol (▵), or epicholesterol (▪). C, diagrams of the OSBP311-809 and OSBPΔ77-310 with the average estimated binding affinity for 25-hydroxycholesterol and cholesterol. A one-way ANOVA followed by multiple comparisons using Fisher's Least-Significant-Difference Test analysis of binding to these constructs is shown in supplemental Fig. S1. D, standard binding protocol was used to measure 25-OH-[3H]cholesterol binding to OSBP311-809 and OSBPΔ77-310. E, in the same experiment we measured [3H]cholesterol binding to OSBP311-809 and OSBPΔ77-310. We used immunoblotting to confirm that each sample within an experiment had the same amount of expressed OSBP constructs.View Large Image Figure ViewerDownload Hi-res image Download (PPT) Regulation of Sterol Binding—With the sterol-binding site in the middle of the molecule it is possible that the C- and N-terminal portions of OSBP are specialized to regulate specific functions of the molecule. Two functions of interest are HePTP/PP2A binding and sterol-binding. Fig. 5A shows that full-length OSBP binds 25-hydroxycholesterol (•). It also binds cholesterol (•, Fig. 5B). The average of multiple trials indicates that in our assay full-length OSBP has a Kd of ∼37 nm for 25-hydroxycholesterol and ∼173 nm for cholesterol (Fig. 5C and supplemental Figs. S1 and S3). Ridgway et al. (15Ridgway N.D. Dawson P.A. Ho Y.K. Brown M.S. Goldstein J.L. J. Cell Biol. 1992; 116: 307-319Crossref PubMed Scopus (241) Google Scholar) reported that OSBP lacking amino acids 455-809 (OSBP1-454), which would delete four amino acids from the C-terminal end of the sterol-binding domain we have localized to OSBP409-459, does not bind oxysterols. We noticed that this set of amino acids contains a tyrosine residue at position 458 within a consensus CRAC motif cholesterol recognition/interaction amino acid consensus: (L/V)X(1-5)YX(1-5)(R/K)). CRAC domains have been implicated in cholesterol binding to peripheral-type benzodiazepine receptor (PBR) (17Jamin N. Neumann J.M. Ostuni M.A. Vu T.K. Yao Z.X. Murail S. Robert J.C. Giatzakis C. Papadopoulos V. Lacapere J.J. Mol. Endocrinol. 2005; 19: 588-594Crossref PubMed Scopus (185) Google Scholar) and Neimann-Pick C2 (18Friedland N. Liou H.L. Lobel P. Stock A.M. Proc. Natl. Acad. Sci. U. S. A. 2003; 100: 2512-2517Crossref PubMed Scopus (260) Google Scholar). Therefore, we constructed an OSBP with an amino acid substitution at position 458 (Y458S) and another with a substitution at 298 (Y298S) within a putative CRAC domain outside of the sterol-binding motif. Each was then tested for oxysterol binding (Fig. 5D). Whereas OSBPY298S exhibited normal, saturable 25-hydroxycholesterol binding with a Kd of ∼15 nm (▪), only nonspecific binding was detected for OSBPY458S (▴). Similar result was obtained for [3H]cholesterol binding (supplemental Fig. S2). These results suggest a role for tyrosine 458 in sterol binding to OSBP. Unlike OSBP408-809 (Fig. 3D), 25-hydroxycholesterol competed poorly for cholesterol binding to full-length OSBP (Fig. 5E). Therefore, we explored the possibility that the N-terminal end of OSBP influenced sterol binding to the sterol-binding site in OSBP408-459. Fig. 6A shows the 25-hydroxycholesterol binding curve for an OSBP that lacks the first 310 amino acids (OSBP311-809, ▪) compared with OSBP1-409 (▴). The estimated Kd for 25-hydroxycholesterol binding to OSBP311-809 was ∼20 nm, but in the same experiment this sterol did not bind OSBP1-409. OSBP311-809 also bound cholesterol (B and E) and binding was competed by both cholesterol (B, •) and 25-hydroxycholesterol (B, ▵) but not epicholesterol (B, ▪). Therefore, the sterol binding characteristics of OSBP are not influenced by the presence of amino acids 311-408 (compare Figs. 6B with 3D). We next constructed an OSBP that had an internal deletion between amino acids 77 and 310 (OSBPΔ77-310, Fig. 6C) and compared the 25-hydroxycholesterol (D) and cholesterol (E) binding characteristic with OSBP311-809. Whereas OSBPΔ77-310 (D, ○) and OSBPΔ77-310 (D, ▴) both bound 25-hydroxycholesterol equally well, cholesterol binding to OSBPΔ77-310 was reduced (E, ▴) compared with OSBP311-809 (E, ○). This suggests that the glycine-alanin" @default.
• W2024692118 created "2016-06-24" @default.
• W2024692118 creator A5033375010 @default.
• W2024692118 creator A5045620483 @default.
• W2024692118 creator A5047635771 @default.
• W2024692118 creator A5070413526 @default.
• W2024692118 date "2008-03-01" @default.
• W2024692118 modified "2023-10-15" @default.
• W2024692118 title "The N Terminus Controls Sterol Binding while the C Terminus Regulates the Scaffolding Function of OSBP" @default.
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