The Processing of Ligands by the Class A Scavenger Receptor Is Dependent on Signal Information Located in the Cytoplasmic Domain
1999; Elsevier BV; Volume: 274; Issue: 51 Linguagem: Inglês
10.1074/jbc.274.51.36808
ISSN1083-351X
Autores Tópico(s)Cholesterol and Lipid Metabolism
ResumoThe mechanisms that regulate the transport of the macrophage class A scavenger receptor during ligand uptake were investigated. Kinetic analysis of the changes in receptor phosphorylation demonstrated that serine phosphorylation increased during the internalization of acetyl-low density lipoproteins (LDL) by macrophages. The increase was maximal at about 2.5 min after the initiation of ligand uptake. Oxidized LDL also stimulated serine phosphorylation, but the relative increase was smaller and the time to maximum was shorter. Receptor mutants expressed in Chinese hamster ovary and COS cells showed that elimination of the potential phosphorylation site at Ser21 increased acetyl-LDL metabolism, whereas inactivation of the site at Ser49reduced acetyl-LDL uptake. The increase in uptake by the Ser21 mutant was due to an increase in surface receptor expression. In contrast, elimination of the site at Ser49did not affect receptor expression but slowed receptor internalization. To identify potential internalization signal sequences, β-turn structure in the cytosolic domain was targeted for mutagenesis. Disruption of one region near Asp25 inhibited receptor activity. The studies support a model whereby receptor internalization requires the presence of an internalization signal motif but that the rate of receptor internalization is governed by the pattern of receptor phosphorylation induced by the ligand. The mechanisms that regulate the transport of the macrophage class A scavenger receptor during ligand uptake were investigated. Kinetic analysis of the changes in receptor phosphorylation demonstrated that serine phosphorylation increased during the internalization of acetyl-low density lipoproteins (LDL) by macrophages. The increase was maximal at about 2.5 min after the initiation of ligand uptake. Oxidized LDL also stimulated serine phosphorylation, but the relative increase was smaller and the time to maximum was shorter. Receptor mutants expressed in Chinese hamster ovary and COS cells showed that elimination of the potential phosphorylation site at Ser21 increased acetyl-LDL metabolism, whereas inactivation of the site at Ser49reduced acetyl-LDL uptake. The increase in uptake by the Ser21 mutant was due to an increase in surface receptor expression. In contrast, elimination of the site at Ser49did not affect receptor expression but slowed receptor internalization. To identify potential internalization signal sequences, β-turn structure in the cytosolic domain was targeted for mutagenesis. Disruption of one region near Asp25 inhibited receptor activity. The studies support a model whereby receptor internalization requires the presence of an internalization signal motif but that the rate of receptor internalization is governed by the pattern of receptor phosphorylation induced by the ligand. low density lipoproteins Dulbecco's modified Eagle's medium fetal calf serum dimethyl sulfoxide chloramphenicol acetyltransferase Chinese hamster ovary Nu-Serum phosphate-buffered saline The predominant feature of the fatty streak lesion of atherosclerosis is the presence of lipid-engorged cells, called foam cells, in the arterial intima. The appearance of fatty streaks in all models of lipid-induced atherosclerosis predicts that they are part of the early events and are important to explore the mechanism by which they are formed. The major cellular events that precede fatty streak formation are fairly consistent between different atherosclerosis models; however, it is clear from the various transgenic and knockout mouse models that the transition to the fatty streak can be initiated by a number of metabolic alterations. This supports the view that it is unlikely that a single pathogenic event accounting for fatty streak lesion formation under all circumstances will be identified, and the net accumulation of cholesterol in the artery wall, in any particular model, will be determined by the balance between the processes that promote cholesterol deposition and those that oppose it. One component of the formulation is likely to be the modified low density lipoprotein (LDL)1-scavenger receptor pathway (reviewed in Refs. 1Steinberg D. Parthasarathy S. Carew T.E. Khoo J.C. Witztum J.L. N. Engl. J. Med. 1989; 320: 915-924Crossref PubMed Google Scholar and 2Steinberg D. J. Biol. 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Morino Y. Takahashi K. Virchow's Arch. B Cell Pathol. Incl. Mol. Pathol. 1986; 52: 1-13Crossref PubMed Scopus (27) Google Scholar). After binding to surface receptors, the receptor-ligand complexes concentrate in coated pits and are rapidly internalized into endosomes. The ligand dissociates from the receptor and is transported within endosomes to lysosomes where it is metabolized while the receptor recycles back to the cell surface. The process is highly efficient and can lead to foam cell formation in vitro. The receptor does not appear to cycle continuously through the metabolic pathway since, in the absence of suitable ligands, receptors are not present in coated pits. This suggests that the process depends upon a stimulus and thus it is regulated. In support of this, pharmacological studies have shown that receptor transport can be modulated. Alterations of calcium flux, protein phosphorylation, or protein glycosylation have been reported to affect scavenger receptor transport (22Fong L.G. J. Lipid Res. 1996; 37: 574-587Abstract Full Text PDF PubMed Google Scholar, 23Bernini F. Scurati N. Bonfadin G. Fumagalli E. Arterioscler. Thromb. Vasc. Biol. 1995; 15: 1352-1358Crossref PubMed Scopus (38) Google Scholar, 24Van Berkel T.J.C. Nagelkerke J.F. Kruijt J.K. FEBS Lett. 1981; 132: 61-66Crossref PubMed Scopus (19) Google Scholar, 25Sulistiyani R.W. St C. Arterioscler. Thromb. Vasc. Biol. 1997; 17: 1691-1700Crossref PubMed Google Scholar). The basis for their actions or the precise step(s) affected has not yet been established. The most direct evidence that receptor transport is tightly controlled is based on the observation of Doi and colleagues (17Doi T. Kurasawa M. Higashino K. Imanishi T. Mori T. Naito M. Takahashi K. Kawabe Y. Wada Y. Matsumoto A. Kodama T. J. Biol. Chem. 1994; 269: 25598-25604Abstract Full Text PDF PubMed Google Scholar). They showed that the substitution of His260 in the α-helical coiled-coil domain of the receptor with a leucine prevents the acid pH-induced dissociation of ligands from the scavenger receptor. When cells expressing this mutant receptor were incubated with ligand, the binding and internalization of ligand proceeded normally; however, after their internalization, the receptors did not recycle back to the cell surface. Recent studies have begun to map regions of the receptor important for ligand binding (26Andersson L. Freeman M.W. J. Biol. Chem. 1998; 273: 19592-19601Abstract Full Text Full Text PDF PubMed Scopus (46) Google Scholar). The present study focuses on the processes likely to control scavenger receptor internalization. There are two of potential importance; the expression of an internalization signal motif and receptor phosphorylation (reviewed in Refs. 27Trowbridge I.S. Collawn J.F. Hopkins C.R. Annu. Rev. Cell Biol. 1993; 9: 129-161Crossref PubMed Scopus (700) Google Scholar and 28Pearse B.M.F. Robinson M.S. Annu. Rev. Cell Biol. 1990; 6: 151-171Crossref PubMed Scopus (534) Google Scholar). The signal motifs are thought to define a three-dimensional conformation and chemistry. These conformations are thought to provide the appropriate signal for the association of the cytoplasmic domain of receptors with components of clathrin-coated pits (28Pearse B.M.F. Robinson M.S. Annu. Rev. Cell Biol. 1990; 6: 151-171Crossref PubMed Scopus (534) Google Scholar, 29Collawn J.F. Stangel M. Kuhn L.A. Esekogwu V. Jing S. Trowbridge I.S. Tainer J.A. Cell. 1990; 63: 1061-1072Abstract Full Text PDF PubMed Scopus (388) Google Scholar, 30Keen J.H. Annu. Rev. Biochem. 1990; 59: 415-438Crossref PubMed Scopus (170) Google Scholar), thereby providing an efficient mechanism for the selective concentration of receptors at sites of internalization. An internalization signal sequence has not been identified for the scavenger receptor; however, the targeting of scavenger receptor-ligand complexes to coated pits strongly suggests that one may be present. Changes in receptor phosphorylation can also modulate receptor internalization. This has been documented for several receptors that are internalized at coated pits including the asialoglycoprotein receptor (31Fallon R.J. Danaher R.L. Saylor R.L. Saxena A. J. Biol. Chem. 1994; 269: 11011-11017Abstract Full Text PDF PubMed Google Scholar), epidermal growth factor receptor (32Wiley H.S. Herbst J.J. Walsh B.J. Lauffenburger D.A. Rosenfeld M.G. Gill G.N. J. Biol. Chem. 1991; 266: 11083-11094Abstract Full Text PDF PubMed Google Scholar), and the insulin receptor (33Carpentier J.L. Paccaud J.P. Baecker J. Gilbert A. Orci L. Kahn C.R. J. Cell Biol. 1993; 122: 1243-1252Crossref PubMed Scopus (49) Google Scholar). The scavenger receptor contains three conserved phosphorylation sites in the cytoplasmic domain of the human, mouse, bovine, and rabbit receptors (34Kodama T. Freeman M. Rohrer L. Zabrecky J. Matsudaira P. Krieger M. Nature. 1990; 343: 531-535Crossref PubMed Scopus (833) Google Scholar, 35Ashkenas J. Penman M. Vasile E. Acton S. Freeman M. Krieger M. J. Lipid Res. 1993; 34: 983-1000Abstract Full Text PDF PubMed Google Scholar, 36Matsumoto A. Naito M. Itakura H. Ikemoto S. Asaoka H. Hayakawa I. Kanamori H. Aburatani H. Takaku F. Suzuki H. Kobari Y. Miyai T. Takahashi K. Cohen E.H. Wydro R. Housman D.E. Kodama T. Proc. Natl. Acad. Sci. U. S. A. 1990; 87: 9133-9137Crossref PubMed Scopus (301) Google Scholar, 37Bickel P.E. Freeman M.W.. J. Clin. Invest. 1992; 90: 1450-1457Crossref PubMed Scopus (106) Google Scholar) located at Ser21, Thr30, and Ser49. We have shown previously that the mouse macrophage scavenger receptor is phosphorylated in situ (22Fong L.G. J. Lipid Res. 1996; 37: 574-587Abstract Full Text PDF PubMed Google Scholar), but the relation of this to receptor transport has not yet been established. This report investigates the role of receptor phosphorylation and potential internalization motifs in the control of receptor function during modified LDL uptake. The results show that the function of the mouse scavenger receptor is dependent on both receptor phosphorylation and one or more internalization signal motifs. The presence of phosphorylation sites in the cytoplasmic domain that affect receptor function both in a positive and negative fashion predict that the level of receptor activity will depend on which sites are phosphorylated during the metabolic cycle. However, the dependence on phosphorylation is not absolute. It is proposed that receptor phosphorylation acts as a modulator of receptor activity by governing the accessibility of one or more internalization signal sequences in the receptor cytoplasmic domain with specific components of clathrin-coated pit structures. Carrier-free Na125I and [14C]chloramphenicol were purchased from Amersham Pharmacia Biotech. Endotoxin tested Dulbecco's modified Eagle's medium (DMEM) and fetal calf serum (FCS) were purchased from Life Technologies, Inc. FCS was heat-inactivated before use (56 °C for 30 min). Nu-Serum (NS) was obtained from Becton Dickenson (Bedford, MA). Butyryl-CoA, chloramphenicol, dimethyl sulfoxide (Me2SO), DEAE-dextran (M r 500,000), chloroquine, phosphoserine, phosphothreonine, and phosphotyrosine were purchased from Sigma. COS-7 and CHO K-1 cells were obtained from American Type Culture Collection (Manassas, VA). Synthetic oligonucleotides were purchased from Genemed Biotechnologies (South San Francisco, CA) or Genosys (Woodlands, TX). Human LDL (d 1.019–1.063 g/ml) was isolated from EDTA-treated plasma by density gradient ultracentrifugation (38Havel R.J. Eder H.A. Bragdon J.H. J. Clin. Invest. 1955; 34: 1345-1353Crossref PubMed Scopus (6449) Google Scholar). LDL was radiolabeled with Na125I using IODO-GEN (Pierce) to specific activities of 200–400 cpm/ng. The acetylation and oxidation of LDL were performed as described previously (18Fong L.G. Fong T.A.T. Cooper A.D. J, Biol, Chem. 1990; 265: 11751-11760Abstract Full Text PDF PubMed Google Scholar, 22Fong L.G. J. Lipid Res. 1996; 37: 574-587Abstract Full Text PDF PubMed Google Scholar). The class A type I mouse scavenger receptor cDNA was kindly provided by Dr. Larry Stanton at Scios Nova (Mountain View, CA). This was subcloned into pcDNA 3.1 (Invitrogen) using the restriction enzyme sites BamHI andNotI. The mutagenesis was performed using the Stratagene QuickchangeTM method that is based on the incorporation of a mutation using Pfu DNA polymerase and complementary mutagenic oligonucleotide primers. The native receptor mutant construct (50 ng) was incubated with 125 ng of each mutagenic oligonucleotide primer (32–38 bases in length) and Pfu DNA polymerase (2.5 units). The temperature cycling parameters followed the recommendations suggested by the manufacturer. The reaction mixture was incubated withDpnI (20 units) to digest the parental DNA template and then used to transform Top10F′ cells (Invitrogen). DNA from selected bacterial colonies was isolated by phenol/chloroform extraction (39Sambrook J. Fritsch E.F. Maniatis T. Molecular Cloning. A Laboratory Manual. 2nd Ed. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY.1989: 1.25-1.28Google Scholar) and analyzed by manual dideoxynucleotide sequencing (Promega Fmol DNA Sequencing System). DNAs containing the correct mutations were identified, and large plasmid preparations were isolated by ion exchange chromatography (Qiagen; Valencia, CA). For a selected number of receptor constructs, the scavenger receptor cDNA was subcloned into the mammalian expression vector pCMV-Tag4 (Stratagene). This produces a fusion protein that contains an extra 10 amino acids (LEDYKDDDDK) located at the carboxyl terminus of the receptor protein. The stop codon in the scavenger receptor cDNA was eliminated and substituted with an XhoI site by polymerase chain reaction using the native scavenger receptor cDNA in pcDNA3.1 as the template and the primers 5′-CAAGCGCACGTGGAACAGGAAGTAAAACAG-3′ and 5′-GATCTCGAGTGAAGTACAAGTGACCCCAGC-3′. The polymerase chain reaction fragment was subcloned into each of the pcDNA 3.1 receptor mutant constructs using the unique restriction enzyme sites Bbr andXhoI. Sequencing confirmed that the epitope DNA sequence was in frame with the receptor cDNA sequence. The entire receptor cDNA was then subcloned into pCMV-Tag4 using the restriction enzyme sites BamHI and XhoI. COS-7 cells were transfected with plasmid DNA using DEAE-dextran (40Aruffo A. Current Protocols in Molecular Biology. 2. Greene Publishing Associates and Wiley-Interscience, New York1991: 16.13.1-16.13.7Google Scholar). COS cells were plated at a density of 4 × 105 cells/10-cm tissue culture dish and incubated overnight in DMEM supplemented with 10% FCS. The following day the cells were washed twice with DMEM containing 10% NS and incubated with the different plasmid DNAs. Prior to their addition to the cells, the DNA preparations were pretreated. The scavenger receptor DNA preparations (7.5 μg) were diluted in Tris-EDTA buffer (20 μl) and added to 15-ml culture tubes. To this was added 6 ml of a freshly prepared stock solution containing 400 μg/ml DEAE-dextran, 0.1 mm chloroquine, and 1.25 μg/ml of the reporter gene construct pcDNA/CAT in DMEM, 10% NS. These were incubated at room temperature for 15 min and then added to the washed cells. After 3.5 h at 37 °C, the cells were incubated with PBS containing 10% Me2SO at room temperature for 2 min. The cells were washed with DMEM, 10% FCS and incubated in the same medium. The next day the cells were dislodged by trypsin treatment, resuspended in 6.5 ml of DMEM, 10% FCS, and added to 4 wells of a 24-well plate (1 ml/well) and 1 well of a 6-well plate (2 ml/well). The metabolism of modified LDL and CAT activities was measured 48 h later. CHO cells were transfected using calcium phosphate (5 Prime → 3 Prime, Inc., Boulder, CO). CHO cells were seeded at a density of 4 × 105 cells/10 cm tissue culture dish and cultured overnight in DMEM/F-12 medium supplemented with 10% FCS. The following day the culture medium was replaced with fresh medium (10 ml/dish), and the cells were incubated for 2 h. The calcium phosphate/DNA precipitate was prepared by incubating plasmid DNA (7.5 μg each of the receptor DNA construct and pcDNA3.1/CAT) with 125 mm CaCl2 and DNA precipitation buffer (25 mm Hepes (pH 7.05), 0.75 mmNa2HPO4, 5 mm KCl, 140 mm NaCl, and 6 mm glucose) in a total volume of 1 ml according to the manufacturer's recommendations. This was added to the cells and incubated together for 4–5 h. The culture medium was removed, and the cells were incubated with 3 ml of 15% glycerol in DNA precipitation buffer at room temperature for 2 min. Phosphate-buffered saline (10 ml/dish) was added and incubated at room temperature for 4 min. The cells were rinsed once with PBS and incubated overnight with fresh tissue culture medium. The next day the cells were plated and analyzed as described above for transfected COS cells. Adherent cells in 24-well plates were rinsed 3 times with DMEM, 10% FCS and incubated with 125I-labeled modified LDL. To measure lipoprotein degradation, the cells were incubated with modified LDL (5 μg/ml) in the same medium (0.5 ml) at 37 °C. The amount of degradation products was determined by measuring the amount of trichloroacetic acid- and silver nitrate (AgNO3)-soluble radioactivity generated in the incubation medium, and the amount of cell associated lipoprotein was determined by measuring the amount of radioactivity associated with an aliquot of a cell lysate (18Fong L.G. Fong T.A.T. Cooper A.D. J, Biol, Chem. 1990; 265: 11751-11760Abstract Full Text PDF PubMed Google Scholar, 22Fong L.G. J. Lipid Res. 1996; 37: 574-587Abstract Full Text PDF PubMed Google Scholar). The amount of specific lipoprotein metabolism (total uptake minus nonspecific uptake) was calculated using fucoidan (100 μg/ml) to determine the amount of nonspecific lipoprotein metabolism. The results are expressed as the amount of degradation or cell-associated products in nanograms per mg cell protein. To measure lipoprotein binding, the cells were incubated with modified LDL (10 μg/ml) in DMEM, 10% FCS buffered with 10 mm Hepes (pH 7.4) at 4 °C for 2 h. Non-bound lipoprotein was removed by washing the cells three times with ice-cold PBS, and the amount bound was determined by measuring the amount of cell-associated radioactivity. The amount of specific binding was calculated and expressed as described above. To correct for differences in transfection efficiencies, the cells were cotransfected with pcDNA/CAT and the results normalized based on the amount of CAT activity (41Kingston R.E. Sheen J. Current Protocols in Molecular Biology. 1. Greene Publishing Associates and Wiley-Interscience, New York1990: 9.6.1-9.6.8Google Scholar). Cells in 6-well culture dishes were washed with PBS and lysed with 200 μl of 0.25m Tris (pH 7.8) containing 0.5% Triton X-100. The cell lysate was transferred to a microcentrifuge tube and stored at −80 °C. The sample was centrifuged for 1 min, and the protein concentration of the supernatant was measured using BCA. An aliquot containing 6 μg of protein for COS cells and 1.5 μg for CHO cells was diluted to a volume of 10 μl with lysis buffer and incubated with an equal volume of substrate mix at 37 °C for 1 h. The substrate mix contained 0.5 mg/ml butyryl-CoA, 0.2 m Tris (pH 8.0), and 4 μCi/ml [14C]chloramphenicol (14.8 mCi/mmol). The reaction mixture was centrifuged briefly in a microcentrifuge and then extracted with 200 μl of xylenes. The xylene layer was transferred to another tube and back-extracted twice with 100 μl of water. The amount of radiolabeled butyrylated chloramphenicol in 80–140 μl of the xylene fraction was then measured by scintillation counting. At these conditions, the conversion of chloramphenicol to butyryl chloramphenicol was between 15 and 40%. Resident mouse peritoneal macrophages in 6-well tissue culture dishes were labeled with [32P]orthophosphate as described previously (22Fong L.G. J. Lipid Res. 1996; 37: 574-587Abstract Full Text PDF PubMed Google Scholar). The cells were washed three times with DMEM, 10% FCS buffered with 10 mm Hepes (pH 7.4) and incubated with unlabeled lipoprotein (50 μg/ml) at 4 °C for 2 h. Non-bound lipoprotein was removed by washing with ice-cold medium and then incubated at 37 °C from 0.5 to 5 min. The cells were immediately placed on ice and washed with ice-cold PBS. Cells that were not incubated at 37 °C but kept on ice were also included to measure protein phosphorylation at time 0. The cells were solubilized, and equivalent amounts of cell lysate protein were incubated with a polyclonal anti-scavenger receptor antibody to precipitate the receptor protein (22Fong L.G. J. Lipid Res. 1996; 37: 574-587Abstract Full Text PDF PubMed Google Scholar). The precipitates were separated by SDS-polyacrylamide gel electrophoresis and transferred to Immobilon P membranes (Millipore) by electroblotting. The location of the receptor was identified by autoradiography, and the piece of membrane containing the immobilized receptor was isolated. The membrane pieces were hydrolyzed in 200 μl of 6 n HCl at 110 °C for 1 h, and the hydrolysate was dried using a speed vacuum. The residue was resuspended in 7 μl of pH 1.9 buffer (H2O/glacial acetic acid/formic acid; 1076/93.6/30 (v/v)) containing 1 mg/ml each of phosphoserine, phosphotyrosine, and phosphothreonine. An aliquot (5 μl) of the suspension was spotted onto thin layer cellulose plates (100 μm; C.B.S. Scientific), and the constituent amino acids were separated using the Hunter Thin Layer Peptide Mapping Electrophoresis System (C.B.S. Scientific) (42Van der Geer P. Luo K. Sefton B.M. Hunter T. Cell Biology, A Laboratory Handbook. 3. Danish Centre for Human Genome Research, Aarhus, Denmark1994: 422-448Google Scholar). Electrophoresis in the first dimension was done at 1.5 kV in pH 1.9 buffer for 20 min and the second dimension at 1.3 kV in pH 3.5 buffer (H2O/glacial acetic acid/pyridine; 1134/60/6 (v/v)) for 16 min. The dried plate was stained with 0.25% ninhydrin to locate the phosphoamino acid standards. The radiolabeled residues were detected using a PhosphorImager (Molecular Dynamics Storm 860), and the amount of radiolabel for each phosphoamino acid was determined using a fixed area and quantified using Image QuaNT software (Molecular Dynamics). Background values from three separate areas were averaged and subtracted from the values measured for each phosphoamino acid. The results were expressed relative to time 0 that was given an arbitrary value of 1. A maximum of four samples can be analyzed on one plate. Thus to compare samples on an equal basis, the number of samples for each experiment was limited to four, and they were analyzed on the same plate. Protein was measured by the method of Lowry (43Lowry O.H. osebrough N.J. Farr A.L. Randall R.J. J. Biol. Chem. 1951; 193: 265-275Abstract Full Text PDF PubMed Google Scholar) or by the Pierce BCA protein assay method (Pierce) using albumin as a standard. Statistical analysis was done using non-paired Student's t test. The cytoplasmic domain of the scavenger receptor A contains three phosphorylation sites that are conserved among all four species for which protein sequence is available. They are located at Ser21, Thr30, and Ser49. The mouse receptor contains a fourth site at Ser36 that is unique to the mouse protein. To examine if receptor phosphorylation and receptor activity might be linked, the level of receptor phosphorylation during the internalization of acetyl-LDL, a high affinity ligand of the scavenger receptor A, by mouse macrophages was measured. To facilitate the detection of changes in phosphorylation, the internalization of surface receptors was synchronized. This would maximize the number of surface receptors that are at the same stage of the internalization process. Cells were incubated with acetyl-LDL at 4 °C to allow surface binding and then washed to remove non-bound lipoprotein. The cells were then incubated at 37 °C to stimulate receptor-ligand internalization, and the level of receptor phosphorylation was monitored by phosphoamino acid analysis. To measure the level of phosphorylation at base line, one set of cells was kept on ice and processed in the same manner. At base line (time 0), there was a low level of receptor phosphorylation on serine residues (Fig. 1). This was present even when cells were preincubated in the absence of lipoprotein (not shown). During the internalization of acetyl-LDL, there was an increase in the phosphorylation of serine residues but no detectable phosphorylation of threonine or tyrosine. The absence of tyrosi
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