SemOpenAlex |

SemOpenAlex

Matches in SemOpenAlex for { <https://semopenalex.org/work/W2048597918> ?p ?o ?g. }

Showing items 1 to 100 of ±116 with 100 items per page.

W2048597918 endingPage "30470" @default.
W2048597918 startingPage "30465" @default.
W2048597918 abstract "The low density lipoprotein (LDL) receptor is a transmembrane glycoprotein performing “receptor-mediated endocytosis” of cholesterol-rich lipoproteins. At the N terminus, the LDL receptor has modular cysteine-rich repeats in both the ligand binding domain and the epidermal growth factor (EGF) precursor homology domain. Each repeat contains six disulfide-bonded cysteine residues, and this structural motif has also been found in many other proteins. The bovine LDL receptor has been purified and reconstituted into egg yolk phosphatidylcholine vesicle bilayers. Using gel electrophoresis and cryoelectron microscopy (cryoEM), the ability of the reconstituted LDL receptor to bind its ligand LDL has been demonstrated. After reduction of the disulfide-bonds in the N-terminal domain of the receptor, the reduced LDL receptor was visualized using cryoEM; reduced LDL receptors showed images with a diffuse density region at the distal end of the extracellular domain. Gold labeling of the reduced cysteine residues was achieved with monomaleimido-Nanogold, and the bound Nanogold was visualized in cryoEM images of the reduced, gold-labeled receptor. Multiple gold particles were observed in the diffuse density region at the distal end of the receptor. Thus, the location of the ligand binding domain of the LDL receptor has been determined, and a model is suggested for the arrangement of the seven cysteine-rich repeats of the ligand binding domain and two EGF-like cysteine-rich repeats of the EGF precursor homology domain. The low density lipoprotein (LDL) receptor is a transmembrane glycoprotein performing “receptor-mediated endocytosis” of cholesterol-rich lipoproteins. At the N terminus, the LDL receptor has modular cysteine-rich repeats in both the ligand binding domain and the epidermal growth factor (EGF) precursor homology domain. Each repeat contains six disulfide-bonded cysteine residues, and this structural motif has also been found in many other proteins. The bovine LDL receptor has been purified and reconstituted into egg yolk phosphatidylcholine vesicle bilayers. Using gel electrophoresis and cryoelectron microscopy (cryoEM), the ability of the reconstituted LDL receptor to bind its ligand LDL has been demonstrated. After reduction of the disulfide-bonds in the N-terminal domain of the receptor, the reduced LDL receptor was visualized using cryoEM; reduced LDL receptors showed images with a diffuse density region at the distal end of the extracellular domain. Gold labeling of the reduced cysteine residues was achieved with monomaleimido-Nanogold, and the bound Nanogold was visualized in cryoEM images of the reduced, gold-labeled receptor. Multiple gold particles were observed in the diffuse density region at the distal end of the receptor. Thus, the location of the ligand binding domain of the LDL receptor has been determined, and a model is suggested for the arrangement of the seven cysteine-rich repeats of the ligand binding domain and two EGF-like cysteine-rich repeats of the EGF precursor homology domain. low density lipoprotein epidermal growth factor cryoelectron microscopy β-migrating very low density lipoprotein apolipoprotein B apolipoprotein E egg yolk phosphatidylcholine 2-mercaptoethylamine chloride The low density lipoprotein (LDL)1 receptor binds cholesterol-rich lipoproteins for receptor-mediated endocytosis and plays a key role in the regulation of cholesterol metabolism. This 115-kDa receptor has a mosaic structure containing five distinguishable domains based originally on the sequence of the human LDL receptor (1Yamamoto T. Davis C.G. Brown M.S. Schneider W.J. Casey M.L. Goldstein J.L. Russell D.W. Cell. 1984; 39: 27-38Abstract Full Text PDF PubMed Scopus (970) Google Scholar); the sequences of LDL receptor from other species also suggest the same domain organization (2Yamamoto T. Bishop R.W. Brown M.S. Goldstein J.L. Russell D.W. Science. 1986; 232: 1230-1237Crossref PubMed Scopus (208) Google Scholar, 3Lee L.Y. Mohler W.A. Schafer B.L. Freudenberger J.S. Byrne-Connolly N. Eager K.B. Mosley S.T. Leighton J.K. Thrift R.N. Davis R.A. Tanaka R.D. Nucleic Acids Res. 1989; 17: 1259-1260Crossref PubMed Scopus (48) Google Scholar, 4Hoffer M.J.V. van Eck M.M. Petrij F. van der Zee A. de Wit E. Meijer D. Grosveld G. Havekes L.M. Hofker M.H. Frants R.R. Biochem. Biophys. Res. Commun. 1993; 191: 880-886Crossref PubMed Scopus (32) Google Scholar, 5Mehta K.D. Chen W.-J. Goldstein J.L. Brown M.S. J. Biol. Chem. 1991; 266: 10406-10414Abstract Full Text PDF PubMed Google Scholar, 6Bishop R.W. J. Lipid Res. 1992; 33: 549-557Abstract Full Text PDF PubMed Google Scholar, 7Russell D.W. Schneider W.J. Yamamoto T. Luskey K.L. Brown M.S. Goldstein J.L. Cell. 1984; 37: 577-585Abstract Full Text PDF PubMed Scopus (215) Google Scholar). These domains, beginning at the N terminus are (i) ligand binding domain, (ii) epidermal growth factor (EGF) precursor homology domain, (iii) O-linked sugar domain, (iv) transmembrane domain, and (v) cytoplasmic domain. The ligand binding domain has the characteristic 7 cysteine-rich repeats consisting of a highly homologous 40-residue amino acid sequence. A study using mutated receptors found that different combinations of the cysteine-rich repeats was responsible for binding to LDL or β-migrating very low density lipoprotein (β-VLDL), both ligands for the LDL receptor (8Russell D.W. Brown M.S. Goldstein J.L. J. Biol. Chem. 1989; 264: 21682-21688Abstract Full Text PDF PubMed Google Scholar). Mutations deleting one of the third to the seventh repeats in the ligand binding domain of the LDL receptor resulted in a marked reduction in LDL binding. β-VLDL binding to the LDL receptor was decreased only by deletion of the fifth cysteine-rich repeat in the ligand binding domain. LDL contains a single high molecular mass (550 kDa) protein, apoB100 (9Knott T.J. Pease R.J. Powell L.M. Wallis S.C. Rall Jr S.C. Innerarity T.L. Blackhart B. Taylor W.H. Marcel Y. Milne R. Johnson D. Fuller M. Lusis A.J. McCarthy B.J. Mahley R.W. Levy-Wilson B. Scott J. Nature. 1986; 323: 734-738Crossref PubMed Scopus (401) Google Scholar), whereas β-VLDL has multiple copies of the smaller (33 kDa) apoE plus one molecule of apoB100 or apoB48 (10Mahley R.W. Innerarity T.L. Rall Jr., S.C. Weisgraber K.H. J. Lipid Res. 1984; 25: 1277-1294Abstract Full Text PDF PubMed Google Scholar). It is thought that the receptor binding domain of apoB100 of LDL may require several of the cysteine-rich repeat modules for efficient binding, whereas the presence of multiple small apoE copies in β-VLDL may allow it to contact with other cysteine-rich repeats of the LDL receptor even when a repeat is deleted.The EGF precursor homology domain has three cysteine-rich “growth factor-like” repeats, two at its N terminus (adjacent to the ligand binding domain) and one at its C terminus (next to the O-glycosylation domain). Deletion analysis in this region of the LDL receptor suggested that the EGF precursor homology domain is required for both efficient binding of LDL and for the acid-dependent conformational change that allows the LDL receptor to release its bound ligand after acidification of the endocytic vesicle (11Davis C.G. Goldstein J.L. Sudhof T.C. Anderson R.G.W. Russell D.W. Brown M.S. Nature. 1987; 326: 760-765Crossref PubMed Scopus (304) Google Scholar). However, the LDL receptor with these deletions was able to bind β-VLDL and to internalize it at a normal rate even though the receptor-recycling rate was slowed. Similar growth factor-like repeats have been identified in many proteins; cell surface receptors, viral proteins, factor IX and X of the blood clotting system, growth factors, and developmental proteins of lower eukaryotes (12Doolittle R.F. Feng D.F. Johnson M.S. Nature. 1984; 307: 558-560Crossref PubMed Scopus (125) Google Scholar, 13Doolittle R.F. Trends Biochem. Sci. 1985; 10: 233-237Abstract Full Text PDF Scopus (143) Google Scholar, 14Sudhof T.C. Russell D.W. Goldstein J.L. Brown M.S. Sanchez-Pescador R. Bell G.I. Science. 1985; 228: 893-895Crossref PubMed Scopus (105) Google Scholar, 15Wharton K.A. Johansen K.M. Xu T. Artavanis-Tsakonas S. Cell. 1985; 43: 567-581Abstract Full Text PDF PubMed Scopus (725) Google Scholar, 16Greenwald I. Cell. 1985; 43: 583-590Abstract Full Text PDF PubMed Scopus (212) Google Scholar, 17Jackman R.W. Beeler D.L. van de Water L. Rosenberg R.D. Proc. Natl. Acad. Sci. U. S. A. 1986; 83: 8834-8838Crossref PubMed Scopus (78) Google Scholar, 18Dahlback B. Lundwall A. Stenflo J. Proc. Natl. Acad. Sci. U. S. A. 1986; 83: 4199-4203Crossref PubMed Scopus (106) Google Scholar). Structural studies of the growth factor-like repeats in different proteins have provided convincing evidence that these cysteine-rich repeat modules are engaged in protein-protein interactions (19Rao Z. Handford P. Mayhew M. Knott V. Brownlee G.G. Stuart D. Cell. 1995; 82: 131-141Abstract Full Text PDF PubMed Scopus (309) Google Scholar).Recently, the atomic resolution structures of the 40-residue cysteine-rich repeats of both the ligand binding and EGF precursor homology domains of the LDL receptor have been determined by NMR spectroscopy and x-ray crystallography. These include the structures of cysteine-rich repeats 1, 2, and 5 in the ligand binding domain (20Daly N.L. Scanlon M.J. Djordjevic J.T. Kroon P.A. Smith R. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 6334-6338Crossref PubMed Scopus (161) Google Scholar, 21Daly N.L. Djordjevic J.T. Kroon P.A. Smith R. Biochemistry. 1995; 34: 14474-14481Crossref PubMed Scopus (83) Google Scholar, 22Fass D. Blacklow S. Kim P.S. Berger J.M. Nature. 1997; 388: 691-693Crossref PubMed Scopus (302) Google Scholar) and the structures of EGF-like modules corresponding to the EGF-like repeats in the EGF precursor homology domain (19Rao Z. Handford P. Mayhew M. Knott V. Brownlee G.G. Stuart D. Cell. 1995; 82: 131-141Abstract Full Text PDF PubMed Scopus (309) Google Scholar, 23Hommel U. Harvey T.S. Driscoll P.C. Campbell I.D. J. Mol. Biol. 1992; 227: 271-282Crossref PubMed Scopus (119) Google Scholar, 24Montelione G.T. Wuthrich K. Burgess A.W. Nice E.C. Wagner G. Gibson K.D. Scheraga H.A. Biochemistry. 1992; 31: 236-249Crossref PubMed Scopus (109) Google Scholar). The crystal structure of the fifth cysteine-rich repeat in the ligand binding domain revealed a hydrophobic ligand binding surface with a well coordinated calcium-binding site (22Fass D. Blacklow S. Kim P.S. Berger J.M. Nature. 1997; 388: 691-693Crossref PubMed Scopus (302) Google Scholar). The disulfide bond linkages were between Cys(I)-Cys(III), Cys(II)-Cys(V), and Cys(IV)-Cys(VI), an identical pattern to that reported for the first and the second repeats (20Daly N.L. Scanlon M.J. Djordjevic J.T. Kroon P.A. Smith R. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 6334-6338Crossref PubMed Scopus (161) Google Scholar, 21Daly N.L. Djordjevic J.T. Kroon P.A. Smith R. Biochemistry. 1995; 34: 14474-14481Crossref PubMed Scopus (83) Google Scholar). The structure of repeat 5 explained why calcium ion is essential for a proper folding and disulfide bond formation of this repeat; furthermore, a sensible rationale for the calcium requirement in ligand binding (25van Driel I.R. Goldstein J.L. Sudhof T.C. Brown M.S. J. Biol. Chem. 1987; 262: 17443-17449Abstract Full Text PDF PubMed Google Scholar) was proposed (26Blacklow S.C. Kim P.S. Nat. Struct. Biol. 1996; 3: 758-762Crossref PubMed Scopus (107) Google Scholar). The structure of a concatamer of repeats 1 and 2 suggests that little or no intermolecular interactions occur between the two modules (27Bieri S. Atkins A.R. Lee H.T. Winzor D.J. Smith R. Kroon P.A. Biochemistry. 1998; 37: 10994-11002Crossref PubMed Scopus (49) Google Scholar), and a recent study of the concatamer of repeats 5 and 6 again indicated that each module is structurally independent of the other (28North C.L. Blacklow S.C. Biochemistry. 1999; 38: 3926-3935Crossref PubMed Scopus (70) Google Scholar).The human EGF is a 53-residue peptide hormone that has a sequence similarity with an EGF-like cysteine-rich repeat in the EGF precursor homology domain of the LDL receptor. The human EGF contains three disulfide-bond cross-links between Cys(I)-Cys(III), Cys(II)-Cys(IV), and Cys(V)-Cys(VI) in conserved order for disulfide-bonds in the EGF-like modules (29Cooke R.M. Wilkinson A.J. Baron M. Pastore A. Tappin M.J. Campbell I.D. Gregory H. Sheard B. Nature. 1987; 327: 339-341Crossref PubMed Scopus (236) Google Scholar). The structure of the EGF-like module in blood clotting factor X in the presence and the absence of calcium showed localized structural changes around the calcium-binding site (30Selander-Sunnerhagen M. Ullner M. Persson E. Teleman O. Stenflo J. Drakenberg T. J. Biol. Chem. 1992; 267: 19642-19649Abstract Full Text PDF PubMed Google Scholar). It is suggested that the calcium-binding site maintains the conformation of the N-terminal region and mediates protein-protein interactions (19Rao Z. Handford P. Mayhew M. Knott V. Brownlee G.G. Stuart D. Cell. 1995; 82: 131-141Abstract Full Text PDF PubMed Scopus (309) Google Scholar).In the accompanying paper (31Jeon H. Shipley G.G. J. Biol. Chem. 2000; 275: 30458-30464Abstract Full Text Full Text PDF PubMed Scopus (22) Google Scholar), stick-like, bent stick-like, and Y-shaped images of frozen, hydrated reconstituted LDL receptors have been observed using cryoelectron microscopy (cryoEM). In this paper, we use reduction of the disulfide linkages, maleimide-Nanogold labeling of the now available cysteine residues, and cryoEM to locate the cysteine-rich ligand binding domain.DISCUSSIONThe functionality of the LDL receptor was confirmed by binding studies to its major ligand, LDL. It has been shown that human LDL binds to the bovine LDL receptor as well as the human LDL receptor (35Schneider W.J. Goldstein J.L. Brown M.S. Methods Enzymol. 1985; 109: 405-417Crossref PubMed Scopus (27) Google Scholar), and for the region that can be compared (the C-terminal ∼25%), their amino acid sequences exhibit very high homology (36Goldstein J.L. Brown M.S. Anderson R.G.W. Russell D.W. Schneider W.J. Annu. Rev. Cell Biol. 1985; 1: 1-39Crossref PubMed Scopus (1106) Google Scholar). This similarity of the human and bovine LDL receptors validates our use of the human LDL for binding studies with the bovine LDL receptor. The detergent-solubilized receptor and the reconstituted receptor showed LDL binding using native gel electrophoresis (Fig. 1) and cryoEM (Fig.2). The existence of some dimer form seen in the native gels supports the earlier report of receptor dimerization (37van Driel I.R. Davis C.G. Goldstein J.L. Brown M.S. J. Biol. Chem. 1987; 262: 16127-16134Abstract Full Text PDF PubMed Google Scholar). The dimer band of the LDL receptor shown by Western blot analysis using the N-terminal antibody seemed to be affected more than the monomer band by increasing the LDL concentration, suggesting that a dimer form could provide higher affinity LDL binding. Whether the dimer form of the LDL receptor exists and functions in vivo remains to be established.Images obtained by cryoEM showed bound LDL particles at the surface of LDL receptor-containing vesicles. Many individual LDL particles were bound to the outside surface of the receptor-containing vesicles, and in some images, the stick-like extracellular domain of the LDL receptor was visible, extending from the membrane surface to the bound LDL particle. Further studies using detergent-solubilized LDL receptor (or expressed LDL receptor domains) may eventually be used to map the location on LDL where the receptor binds; a similar approach has been used to detect antibody binding sites on the surface of LDL (38Spin J. Cryoelectron Microscopy Studies of Low Density Lipoprotein in Vitreous Ice. Ph.D. thesis. Boston University, 1997Google Scholar).The successful application of monomaleimido-Nanogold labeling methods in our structural studies of the LDL receptor (31Jeon H. Shipley G.G. J. Biol. Chem. 2000; 275: 30458-30464Abstract Full Text Full Text PDF PubMed Scopus (22) Google Scholar) encouraged us to use additional cysteine residues for further labeling, particularly those of the cysteine-rich N-terminal region. The reduction of these disulfide bonds was performed carefully so as not to perturb the whole structure of the LDL receptor. The LDL receptor bands that were detected in LI silver stain, silver stain, and Western blot analyses after MEA disulfide reduction were normal in appearance. Images of the reduced LDL receptor obtained by cryoEM showed more diffuse density at the end of the extracellular domain. Some images of the reduced LDL receptor still had visible stick-like or Y-shaped density as the previously reported images of the reconstituted receptor (31Jeon H. Shipley G.G. J. Biol. Chem. 2000; 275: 30458-30464Abstract Full Text Full Text PDF PubMed Scopus (22) Google Scholar). The distal end of both the stick-like receptor and the Y-shaped receptor images showed additional diffuse density.CryoEM of reduced, Nanogold-labeled LDL receptor localized the N-terminal cysteine-rich repeats to the distal end of the observed receptor images. There were significant differences in the number of Nanogold particles bound in this distal region, presumably reflecting differences in the number of accessible free cysteine residues and their Nanogold labeling. It seems reasonable to conclude that this distal domain of the imaged LDL receptor contains the seven cysteine-rich repeats of the ligand binding domain plus the two N-terminal cysteine-rich repeats of the EGF precursor homology domain.Studies of repeat motifs common to the human LDL receptor, human EGF, and other proteins have provided structural information relevant to our studies of the bovine LDL receptor. The seven cysteine-rich repeats in the ligand binding domain of the human LDL receptor sequence are connected to each other with 4-residue linker sequences, with the exception of a 12-residue sequence connecting the fourth and the fifth repeats. Two EGF-like cysteine-rich repeats of the EGF precursor homology domain closely follow the seventh repeat of the ligand binding domain. This proximity in the sequence and the proposed function of the EGF-like module in protein-protein interactions suggests that both EGF-like repeats are located near the ligand binding domain at the N terminus of the LDL receptor. The spacer sequence of the EGF precursor homology domain occupies about 70% of this 400-amino acid domain and connects the second and the third cysteine-rich EGF-like repeats. The relatively short (58 amino acids) O-linked sugar domain has been suggested to function as a strut, extending from the membrane surface (36Goldstein J.L. Brown M.S. Anderson R.G.W. Russell D.W. Schneider W.J. Annu. Rev. Cell Biol. 1985; 1: 1-39Crossref PubMed Scopus (1106) Google Scholar), and it seems reasonable that the major part of the EGF precursor homology domain essentially extends this strut to the cysteine-rich domain.In Fig. 6 we suggest possible arrangements, particularly for the cysteine-rich repeat modules of both the ligand binding domain and the EGF precursor homology domain, consistent with the observed cryoEM images of both unlabeled and gold-labeled LDL receptor. Two issues led us to the proposed (2 + 5)-type and (4 + 3)-type arrangements of the 7 cysteine-rich repeat modules of the ligand binding domain. First, for the (2 + 5) type, binding of LDL was severely affected by mutations in repeats 3 to 7, which might imply that the first and the second repeats were located away from the binding site for the LDL (see Fig. 6 A). Second, for the (4 + 3) type, the presence of a longer linker sequence between repeats 4 and 5 suggests that a turn may occur after the fourth repeat in the ligand binding domain (see Fig. 6 B). Since the biological function of the repeated EGF-like modules in protein-protein interaction is significantly calcium-dependent, it has been suggested that an adjacent pair of this module could act as a functional unit (39Rebay I. Fleming R.J. Fehon R.G. Cherbas L. Cherbas P. Artavanis-Tsakonas S. Cell. 1991; 67: 687-699Abstract Full Text PDF PubMed Scopus (596) Google Scholar). Therefore, the two EGF-like repeats were positioned as a pair in the model (see Fig. 6, A and B). Finally, we have suggested possible arrangements for dimeric assemblies of the LDL receptor based on these (2 + 5) and (4 + 3) structural motifs (Fig. 6, right).In summary, the LDL receptor is a single chain membrane receptor that requires detergents or lipids for solubilization. This characteristic of the LDL receptor has made it difficult to investigate its detailed structure by x-ray crystallography. Our cryoEM approach has provided low resolution images of the full-length bovine LDL receptor (31Jeon H. Shipley G.G. J. Biol. Chem. 2000; 275: 30458-30464Abstract Full Text Full Text PDF PubMed Scopus (22) Google Scholar), and in this study we have used the gold labeling approach to determine the location of the cysteine-rich ligand binding domain. However, more detailed structural information must await improvements in specimen preparation for cryoEM and the crystallization of progressively larger extracellular domains of the LDL receptor (40Dirlam K.A. Gretch D.G. LaCount D.J. Sturley S.L. Attie A.D. Protein Expression Purif. 1996; 8: 489-500Crossref PubMed Scopus (21) Google Scholar, 41Simmons T. Newhouse Y.M. Arnold K.S. Innerarity T.L. Weisgraber K.H. J. Biol. Chem. 1997; 272: 25531-25536Abstract Full Text Full Text PDF PubMed Scopus (38) Google Scholar). The low density lipoprotein (LDL)1 receptor binds cholesterol-rich lipoproteins for receptor-mediated endocytosis and plays a key role in the regulation of cholesterol metabolism. This 115-kDa receptor has a mosaic structure containing five distinguishable domains based originally on the sequence of the human LDL receptor (1Yamamoto T. Davis C.G. Brown M.S. Schneider W.J. Casey M.L. Goldstein J.L. Russell D.W. Cell. 1984; 39: 27-38Abstract Full Text PDF PubMed Scopus (970) Google Scholar); the sequences of LDL receptor from other species also suggest the same domain organization (2Yamamoto T. Bishop R.W. Brown M.S. Goldstein J.L. Russell D.W. Science. 1986; 232: 1230-1237Crossref PubMed Scopus (208) Google Scholar, 3Lee L.Y. Mohler W.A. Schafer B.L. Freudenberger J.S. Byrne-Connolly N. Eager K.B. Mosley S.T. Leighton J.K. Thrift R.N. Davis R.A. Tanaka R.D. Nucleic Acids Res. 1989; 17: 1259-1260Crossref PubMed Scopus (48) Google Scholar, 4Hoffer M.J.V. van Eck M.M. Petrij F. van der Zee A. de Wit E. Meijer D. Grosveld G. Havekes L.M. Hofker M.H. Frants R.R. Biochem. Biophys. Res. Commun. 1993; 191: 880-886Crossref PubMed Scopus (32) Google Scholar, 5Mehta K.D. Chen W.-J. Goldstein J.L. Brown M.S. J. Biol. Chem. 1991; 266: 10406-10414Abstract Full Text PDF PubMed Google Scholar, 6Bishop R.W. J. Lipid Res. 1992; 33: 549-557Abstract Full Text PDF PubMed Google Scholar, 7Russell D.W. Schneider W.J. Yamamoto T. Luskey K.L. Brown M.S. Goldstein J.L. Cell. 1984; 37: 577-585Abstract Full Text PDF PubMed Scopus (215) Google Scholar). These domains, beginning at the N terminus are (i) ligand binding domain, (ii) epidermal growth factor (EGF) precursor homology domain, (iii) O-linked sugar domain, (iv) transmembrane domain, and (v) cytoplasmic domain. The ligand binding domain has the characteristic 7 cysteine-rich repeats consisting of a highly homologous 40-residue amino acid sequence. A study using mutated receptors found that different combinations of the cysteine-rich repeats was responsible for binding to LDL or β-migrating very low density lipoprotein (β-VLDL), both ligands for the LDL receptor (8Russell D.W. Brown M.S. Goldstein J.L. J. Biol. Chem. 1989; 264: 21682-21688Abstract Full Text PDF PubMed Google Scholar). Mutations deleting one of the third to the seventh repeats in the ligand binding domain of the LDL receptor resulted in a marked reduction in LDL binding. β-VLDL binding to the LDL receptor was decreased only by deletion of the fifth cysteine-rich repeat in the ligand binding domain. LDL contains a single high molecular mass (550 kDa) protein, apoB100 (9Knott T.J. Pease R.J. Powell L.M. Wallis S.C. Rall Jr S.C. Innerarity T.L. Blackhart B. Taylor W.H. Marcel Y. Milne R. Johnson D. Fuller M. Lusis A.J. McCarthy B.J. Mahley R.W. Levy-Wilson B. Scott J. Nature. 1986; 323: 734-738Crossref PubMed Scopus (401) Google Scholar), whereas β-VLDL has multiple copies of the smaller (33 kDa) apoE plus one molecule of apoB100 or apoB48 (10Mahley R.W. Innerarity T.L. Rall Jr., S.C. Weisgraber K.H. J. Lipid Res. 1984; 25: 1277-1294Abstract Full Text PDF PubMed Google Scholar). It is thought that the receptor binding domain of apoB100 of LDL may require several of the cysteine-rich repeat modules for efficient binding, whereas the presence of multiple small apoE copies in β-VLDL may allow it to contact with other cysteine-rich repeats of the LDL receptor even when a repeat is deleted. The EGF precursor homology domain has three cysteine-rich “growth factor-like” repeats, two at its N terminus (adjacent to the ligand binding domain) and one at its C terminus (next to the O-glycosylation domain). Deletion analysis in this region of the LDL receptor suggested that the EGF precursor homology domain is required for both efficient binding of LDL and for the acid-dependent conformational change that allows the LDL receptor to release its bound ligand after acidification of the endocytic vesicle (11Davis C.G. Goldstein J.L. Sudhof T.C. Anderson R.G.W. Russell D.W. Brown M.S. Nature. 1987; 326: 760-765Crossref PubMed Scopus (304) Google Scholar). However, the LDL receptor with these deletions was able to bind β-VLDL and to internalize it at a normal rate even though the receptor-recycling rate was slowed. Similar growth factor-like repeats have been identified in many proteins; cell surface receptors, viral proteins, factor IX and X of the blood clotting system, growth factors, and developmental proteins of lower eukaryotes (12Doolittle R.F. Feng D.F. Johnson M.S. Nature. 1984; 307: 558-560Crossref PubMed Scopus (125) Google Scholar, 13Doolittle R.F. Trends Biochem. Sci. 1985; 10: 233-237Abstract Full Text PDF Scopus (143) Google Scholar, 14Sudhof T.C. Russell D.W. Goldstein J.L. Brown M.S. Sanchez-Pescador R. Bell G.I. Science. 1985; 228: 893-895Crossref PubMed Scopus (105) Google Scholar, 15Wharton K.A. Johansen K.M. Xu T. Artavanis-Tsakonas S. Cell. 1985; 43: 567-581Abstract Full Text PDF PubMed Scopus (725) Google Scholar, 16Greenwald I. Cell. 1985; 43: 583-590Abstract Full Text PDF PubMed Scopus (212) Google Scholar, 17Jackman R.W. Beeler D.L. van de Water L. Rosenberg R.D. Proc. Natl. Acad. Sci. U. S. A. 1986; 83: 8834-8838Crossref PubMed Scopus (78) Google Scholar, 18Dahlback B. Lundwall A. Stenflo J. Proc. Natl. Acad. Sci. U. S. A. 1986; 83: 4199-4203Crossref PubMed Scopus (106) Google Scholar). Structural studies of the growth factor-like repeats in different proteins have provided convincing evidence that these cysteine-rich repeat modules are engaged in protein-protein interactions (19Rao Z. Handford P. Mayhew M. Knott V. Brownlee G.G. Stuart D. Cell. 1995; 82: 131-141Abstract Full Text PDF PubMed Scopus (309) Google Scholar). Recently, the atomic resolution structures of the 40-residue cysteine-rich repeats of both the ligand binding and EGF precursor homology domains of the LDL receptor have been determined by NMR spectroscopy and x-ray crystallography. These include the structures of cysteine-rich repeats 1, 2, and 5 in the ligand binding domain (20Daly N.L. Scanlon M.J. Djordjevic J.T. Kroon P.A. Smith R. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 6334-6338Crossref PubMed Scopus (161) Google Scholar, 21Daly N.L. Djordjevic J.T. Kroon P.A. Smith R. Biochemistry. 1995; 34: 14474-14481Crossref PubMed Scopus (83) Google Scholar, 22Fass D. Blacklow S. Kim P.S. Berger J.M. Nature. 1997; 388: 691-693Crossref PubMed Scopus (302) Google Scholar) and the structures of EGF-like modules corresponding to the EGF-like repeats in the EGF precursor homology domain (19Rao Z. Handford P. Mayhew M. Knott V. Brownlee G.G. Stuart D. Cell. 1995; 82: 131-141Abstract Full Text PDF PubMed Scopus (309) Google Scholar, 23Hommel U. Harvey T.S. Driscoll P.C. Campbell I.D. J. Mol. Biol. 1992; 227: 271-282Crossref PubMed Scopus (119) Google Scholar, 24Montelione G.T. Wuthrich K. Burgess A.W. Nice E.C. Wagner G. Gibson K.D. Scheraga H.A. Biochemistry. 1992; 31: 236-249Crossref PubMed Scopus (109) Google Scholar). The crystal structure of the fifth cysteine-rich repeat in the ligand binding domain revealed a hydrophobic ligand binding surface with a well coordinated calcium-binding site (22Fass D. Blacklow S. Kim P.S. Berger J.M. Nature. 1997; 388: 691-693Crossref PubMed Scopus (302) Google Scholar). The disulfide bond linkages were between Cys(I)-Cys(III), Cys(II)-Cys(V), and Cys(IV)-Cys(VI), an identical pattern to that reported for the first and the second repeats (20Daly N.L. Scanlon M.J. Djordjevic J.T. Kroon P.A. Smith R. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 6334-6338Crossref PubMed Scopus (161) Google Scholar, 21Daly N.L. Djordjevic J.T. Kroon P.A. Smith R. Biochemistry. 1995; 34: 14474-14481Crossref PubMed Scopus (83) Google Scholar). The structure of repeat 5 explained why calcium ion is essential for a proper folding and disulfide bond formation of this repeat; furthermore, a sensible rationale for the calcium requirement in ligand binding (25van Driel I.R. Goldstein J.L. Sudhof T.C. Brown M.S. J. Biol. Chem. 1987; 262: 17443-17449Abstract Full Text PDF PubMed Google Scholar) was proposed (26Blacklow S.C. Kim P.S. Nat. Struct. Biol. 1996; 3: 758-762Crossref PubMed Scopus (107) Google Scholar). The structure of a concatamer of repeats 1 and 2 suggests that little or no intermolecular interactions occur between the two modules (27Bieri S. Atkins A.R. Lee H.T. Winzor D.J. Smith R. Kroon P.A. Biochemistry. 1998; 37: 10994-11002Crossref PubMed Scopus (49) Google Scholar), and a recent study of the concatamer of repeats 5 and 6 again indicated that each module is structurally independent of the other (28North C.L. Blacklow S.C. Biochemistry. 1999; 38: 3926-3935Crossref PubMed Scopus (70) Google Scholar). The human EGF is a 53-residue peptide hormone that has a sequence similarity with an EGF-like cysteine-rich repeat in the EGF precursor homology domain of the LDL receptor. The human EGF contains three disulfide-bond cross-links between Cys(I)-Cys(III), Cys(II)-Cys(IV), and Cys(V)-Cys(VI) in conserved order for disulfide-bonds in the EGF-like modules (29Cooke R.M. Wilkinson A.J. Baron M. Pastore A. Tappin M.J. Campbell I.D. Gregory H. Sheard B. Nature. 1987; 327: 339-341Crossref PubMed Scopus (236) Google Scholar). The structure of the EGF-like module in blood clotting factor X in the presence and the absence of calcium showed localized structural changes around the calcium-binding site (30Selander-Sunnerhagen M. Ullner M. Persson E. Teleman O. Stenflo J. Drakenberg T. J. Biol. Chem. 1992; 267: 19642-19649Abstract Full Text PDF PubMed Google Scholar). It is suggested that the calcium-binding site maintains the conformation of the N-terminal region and mediates protein-protein interactions (19Rao Z. Handford P. Mayhew M. Knott V. Brownlee G.G. Stuart D. Cell. 1995; 82: 131-141Abstract Full Text PDF PubMed Scopus (309) Google Scholar). In the accompanying paper (31Jeon H. Shipley G.G. J. Biol. Chem. 2000; 275: 30458-30464Abstract Full Text Full Text PDF PubMed Scopus (22) Google Scholar), stick-like, bent stick-like, and Y-shaped images of frozen, hydrated reconstituted LDL receptors have been observed using cryoelectron microscopy (cryoEM). In this paper, we use reduction of the disulfide linkages, maleimide-Nanogold labeling of the now available cysteine residues, and cryoEM to locate the cysteine-rich ligand binding domain. DISCUSSIONThe functionality of the LDL receptor was confirmed by binding studies to its major ligand, LDL. It has been shown that human LDL binds to the bovine LDL receptor as well as the human LDL receptor (35Schneider W.J. Goldstein J.L. Brown M.S. Methods Enzymol. 1985; 109: 405-417Crossref PubMed Scopus (27) Google Scholar), and for the region that can be compared (the C-terminal ∼25%), their amino acid sequences exhibit very high homology (36Goldstein J.L. Brown M.S. Anderson R.G.W. Russell D.W. Schneider W.J. Annu. Rev. Cell Biol. 1985; 1: 1-39Crossref PubMed Scopus (1106) Google Scholar). This similarity of the human and bovine LDL receptors validates our use of the human LDL for binding studies with the bovine LDL receptor. The detergent-solubilized receptor and the reconstituted receptor showed LDL binding using native gel electrophoresis (Fig. 1) and cryoEM (Fig.2). The existence of some dimer form seen in the native gels supports the earlier report of receptor dimerization (37van Driel I.R. Davis C.G. Goldstein J.L. Brown M.S. J. Biol. Chem. 1987; 262: 16127-16134Abstract Full Text PDF PubMed Google Scholar). The dimer band of the LDL receptor shown by Western blot analysis using the N-terminal antibody seemed to be affected more than the monomer band by increasing the LDL concentration, suggesting that a dimer form could provide higher affinity LDL binding. Whether the dimer form of the LDL receptor exists and functions in vivo remains to be established.Images obtained by cryoEM showed bound LDL particles at the surface of LDL receptor-containing vesicles. Many individual LDL particles were bound to the outside surface of the receptor-containing vesicles, and in some images, the stick-like extracellular domain of the LDL receptor was visible, extending from the membrane surface to the bound LDL particle. Further studies using detergent-solubilized LDL receptor (or expressed LDL receptor domains) may eventually be used to map the location on LDL where the receptor binds; a similar approach has been used to detect antibody binding sites on the surface of LDL (38Spin J. Cryoelectron Microscopy Studies of Low Density Lipoprotein in Vitreous Ice. Ph.D. thesis. Boston University, 1997Google Scholar).The successful application of monomaleimido-Nanogold labeling methods in our structural studies of the LDL receptor (31Jeon H. Shipley G.G. J. Biol. Chem. 2000; 275: 30458-30464Abstract Full Text Full Text PDF PubMed Scopus (22) Google Scholar) encouraged us to use additional cysteine residues for further labeling, particularly those of the cysteine-rich N-terminal region. The reduction of these disulfide bonds was performed carefully so as not to perturb the whole structure of the LDL receptor. The LDL receptor bands that were detected in LI silver stain, silver stain, and Western blot analyses after MEA disulfide reduction were normal in appearance. Images of the reduced LDL receptor obtained by cryoEM showed more diffuse density at the end of the extracellular domain. Some images of the reduced LDL receptor still had visible stick-like or Y-shaped density as the previously reported images of the reconstituted receptor (31Jeon H. Shipley G.G. J. Biol. Chem. 2000; 275: 30458-30464Abstract Full Text Full Text PDF PubMed Scopus (22) Google Scholar). The distal end of both the stick-like receptor and the Y-shaped receptor images showed additional diffuse density.CryoEM of reduced, Nanogold-labeled LDL receptor localized the N-terminal cysteine-rich repeats to the distal end of the observed receptor images. There were significant differences in the number of Nanogold particles bound in this distal region, presumably reflecting differences in the number of accessible free cysteine residues and their Nanogold labeling. It seems reasonable to conclude that this distal domain of the imaged LDL receptor contains the seven cysteine-rich repeats of the ligand binding domain plus the two N-terminal cysteine-rich repeats of the EGF precursor homology domain.Studies of repeat motifs common to the human LDL receptor, human EGF, and other proteins have provided structural information relevant to our studies of the bovine LDL receptor. The seven cysteine-rich repeats in the ligand binding domain of the human LDL receptor sequence are connected to each other with 4-residue linker sequences, with the exception of a 12-residue sequence connecting the fourth and the fifth repeats. Two EGF-like cysteine-rich repeats of the EGF precursor homology domain closely follow the seventh repeat of the ligand binding domain. This proximity in the sequence and the proposed function of the EGF-like module in protein-protein interactions suggests that both EGF-like repeats are located near the ligand binding domain at the N terminus of the LDL receptor. The spacer sequence of the EGF precursor homology domain occupies about 70% of this 400-amino acid domain and connects the second and the third cysteine-rich EGF-like repeats. The relatively short (58 amino acids) O-linked sugar domain has been suggested to function as a strut, extending from the membrane surface (36Goldstein J.L. Brown M.S. Anderson R.G.W. Russell D.W. Schneider W.J. Annu. Rev. Cell Biol. 1985; 1: 1-39Crossref PubMed Scopus (1106) Google Scholar), and it seems reasonable that the major part of the EGF precursor homology domain essentially extends this strut to the cysteine-rich domain.In Fig. 6 we suggest possible arrangements, particularly for the cysteine-rich repeat modules of both the ligand binding domain and the EGF precursor homology domain, consistent with the observed cryoEM images of both unlabeled and gold-labeled LDL receptor. Two issues led us to the proposed (2 + 5)-type and (4 + 3)-type arrangements of the 7 cysteine-rich repeat modules of the ligand binding domain. First, for the (2 + 5) type, binding of LDL was severely affected by mutations in repeats 3 to 7, which might imply that the first and the second repeats were located away from the binding site for the LDL (see Fig. 6 A). Second, for the (4 + 3) type, the presence of a longer linker sequence between repeats 4 and 5 suggests that a turn may occur after the fourth repeat in the ligand binding domain (see Fig. 6 B). Since the biological function of the repeated EGF-like modules in protein-protein interaction is significantly calcium-dependent, it has been suggested that an adjacent pair of this module could act as a functional unit (39Rebay I. Fleming R.J. Fehon R.G. Cherbas L. Cherbas P. Artavanis-Tsakonas S. Cell. 1991; 67: 687-699Abstract Full Text PDF PubMed Scopus (596) Google Scholar). Therefore, the two EGF-like repeats were positioned as a pair in the model (see Fig. 6, A and B). Finally, we have suggested possible arrangements for dimeric assemblies of the LDL receptor based on these (2 + 5) and (4 + 3) structural motifs (Fig. 6, right).In summary, the LDL receptor is a single chain membrane receptor that requires detergents or lipids for solubilization. This characteristic of the LDL receptor has made it difficult to investigate its detailed structure by x-ray crystallography. Our cryoEM approach has provided low resolution images of the full-length bovine LDL receptor (31Jeon H. Shipley G.G. J. Biol. Chem. 2000; 275: 30458-30464Abstract Full Text Full Text PDF PubMed Scopus (22) Google Scholar), and in this study we have used the gold labeling approach to determine the location of the cysteine-rich ligand binding domain. However, more detailed structural information must await improvements in specimen preparation for cryoEM and the crystallization of progressively larger extracellular domains of the LDL receptor (40Dirlam K.A. Gretch D.G. LaCount D.J. Sturley S.L. Attie A.D. Protein Expression Purif. 1996; 8: 489-500Crossref PubMed Scopus (21) Google Scholar, 41Simmons T. Newhouse Y.M. Arnold K.S. Innerarity T.L. Weisgraber K.H. J. Biol. Chem. 1997; 272: 25531-25536Abstract Full Text Full Text PDF PubMed Scopus (38) Google Scholar). The functionality of the LDL receptor was confirmed by binding studies to its major ligand, LDL. It has been shown that human LDL binds to the bovine LDL receptor as well as the human LDL receptor (35Schneider W.J. Goldstein J.L. Brown M.S. Methods Enzymol. 1985; 109: 405-417Crossref PubMed Scopus (27) Google Scholar), and for the region that can be compared (the C-terminal ∼25%), their amino acid sequences exhibit very high homology (36Goldstein J.L. Brown M.S. Anderson R.G.W. Russell D.W. Schneider W.J. Annu. Rev. Cell Biol. 1985; 1: 1-39Crossref PubMed Scopus (1106) Google Scholar). This similarity of the human and bovine LDL receptors validates our use of the human LDL for binding studies with the bovine LDL receptor. The detergent-solubilized receptor and the reconstituted receptor showed LDL binding using native gel electrophoresis (Fig. 1) and cryoEM (Fig.2). The existence of some dimer form seen in the native gels supports the earlier report of receptor dimerization (37van Driel I.R. Davis C.G. Goldstein J.L. Brown M.S. J. Biol. Chem. 1987; 262: 16127-16134Abstract Full Text PDF PubMed Google Scholar). The dimer band of the LDL receptor shown by Western blot analysis using the N-terminal antibody seemed to be affected more than the monomer band by increasing the LDL concentration, suggesting that a dimer form could provide higher affinity LDL binding. Whether the dimer form of the LDL receptor exists and functions in vivo remains to be established. Images obtained by cryoEM showed bound LDL particles at the surface of LDL receptor-containing vesicles. Many individual LDL particles were bound to the outside surface of the receptor-containing vesicles, and in some images, the stick-like extracellular domain of the LDL receptor was visible, extending from the membrane surface to the bound LDL particle. Further studies using detergent-solubilized LDL receptor (or expressed LDL receptor domains) may eventually be used to map the location on LDL where the receptor binds; a similar approach has been used to detect antibody binding sites on the surface of LDL (38Spin J. Cryoelectron Microscopy Studies of Low Density Lipoprotein in Vitreous Ice. Ph.D. thesis. Boston University, 1997Google Scholar). The successful application of monomaleimido-Nanogold labeling methods in our structural studies of the LDL receptor (31Jeon H. Shipley G.G. J. Biol. Chem. 2000; 275: 30458-30464Abstract Full Text Full Text PDF PubMed Scopus (22) Google Scholar) encouraged us to use additional cysteine residues for further labeling, particularly those of the cysteine-rich N-terminal region. The reduction of these disulfide bonds was performed carefully so as not to perturb the whole structure of the LDL receptor. The LDL receptor bands that were detected in LI silver stain, silver stain, and Western blot analyses after MEA disulfide reduction were normal in appearance. Images of the reduced LDL receptor obtained by cryoEM showed more diffuse density at the end of the extracellular domain. Some images of the reduced LDL receptor still had visible stick-like or Y-shaped density as the previously reported images of the reconstituted receptor (31Jeon H. Shipley G.G. J. Biol. Chem. 2000; 275: 30458-30464Abstract Full Text Full Text PDF PubMed Scopus (22) Google Scholar). The distal end of both the stick-like receptor and the Y-shaped receptor images showed additional diffuse density. CryoEM of reduced, Nanogold-labeled LDL receptor localized the N-terminal cysteine-rich repeats to the distal end of the observed receptor images. There were significant differences in the number of Nanogold particles bound in this distal region, presumably reflecting differences in the number of accessible free cysteine residues and their Nanogold labeling. It seems reasonable to conclude that this distal domain of the imaged LDL receptor contains the seven cysteine-rich repeats of the ligand binding domain plus the two N-terminal cysteine-rich repeats of the EGF precursor homology domain. Studies of repeat motifs common to the human LDL receptor, human EGF, and other proteins have provided structural information relevant to our studies of the bovine LDL receptor. The seven cysteine-rich repeats in the ligand binding domain of the human LDL receptor sequence are connected to each other with 4-residue linker sequences, with the exception of a 12-residue sequence connecting the fourth and the fifth repeats. Two EGF-like cysteine-rich repeats of the EGF precursor homology domain closely follow the seventh repeat of the ligand binding domain. This proximity in the sequence and the proposed function of the EGF-like module in protein-protein interactions suggests that both EGF-like repeats are located near the ligand binding domain at the N terminus of the LDL receptor. The spacer sequence of the EGF precursor homology domain occupies about 70% of this 400-amino acid domain and connects the second and the third cysteine-rich EGF-like repeats. The relatively short (58 amino acids) O-linked sugar domain has been suggested to function as a strut, extending from the membrane surface (36Goldstein J.L. Brown M.S. Anderson R.G.W. Russell D.W. Schneider W.J. Annu. Rev. Cell Biol. 1985; 1: 1-39Crossref PubMed Scopus (1106) Google Scholar), and it seems reasonable that the major part of the EGF precursor homology domain essentially extends this strut to the cysteine-rich domain. In Fig. 6 we suggest possible arrangements, particularly for the cysteine-rich repeat modules of both the ligand binding domain and the EGF precursor homology domain, consistent with the observed cryoEM images of both unlabeled and gold-labeled LDL receptor. Two issues led us to the proposed (2 + 5)-type and (4 + 3)-type arrangements of the 7 cysteine-rich repeat modules of the ligand binding domain. First, for the (2 + 5) type, binding of LDL was severely affected by mutations in repeats 3 to 7, which might imply that the first and the second repeats were located away from the binding site for the LDL (see Fig. 6 A). Second, for the (4 + 3) type, the presence of a longer linker sequence between repeats 4 and 5 suggests that a turn may occur after the fourth repeat in the ligand binding domain (see Fig. 6 B). Since the biological function of the repeated EGF-like modules in protein-protein interaction is significantly calcium-dependent, it has been suggested that an adjacent pair of this module could act as a functional unit (39Rebay I. Fleming R.J. Fehon R.G. Cherbas L. Cherbas P. Artavanis-Tsakonas S. Cell. 1991; 67: 687-699Abstract Full Text PDF PubMed Scopus (596) Google Scholar). Therefore, the two EGF-like repeats were positioned as a pair in the model (see Fig. 6, A and B). Finally, we have suggested possible arrangements for dimeric assemblies of the LDL receptor based on these (2 + 5) and (4 + 3) structural motifs (Fig. 6, right). In summary, the LDL receptor is a single chain membrane receptor that requires detergents or lipids for solubilization. This characteristic of the LDL receptor has made it difficult to investigate its detailed structure by x-ray crystallography. Our cryoEM approach has provided low resolution images of the full-length bovine LDL receptor (31Jeon H. Shipley G.G. J. Biol. Chem. 2000; 275: 30458-30464Abstract Full Text Full Text PDF PubMed Scopus (22) Google Scholar), and in this study we have used the gold labeling approach to determine the location of the cysteine-rich ligand binding domain. However, more detailed structural information must await improvements in specimen preparation for cryoEM and the crystallization of progressively larger extracellular domains of the LDL receptor (40Dirlam K.A. Gretch D.G. LaCount D.J. Sturley S.L. Attie A.D. Protein Expression Purif. 1996; 8: 489-500Crossref PubMed Scopus (21) Google Scholar, 41Simmons T. Newhouse Y.M. Arnold K.S. Innerarity T.L. Weisgraber K.H. J. Biol. Chem. 1997; 272: 25531-25536Abstract Full Text Full Text PDF PubMed Scopus (38) Google Scholar). We thank Drs. David Atkinson, Esther Bullitt, Kumkum Saxena, and Christine Woldin for helpful advice. Technical assistance was provided by Ann Tercyak, Cheryl England, Michael Gigliotti, Cynthia Curry, and Donald Gantz. Also, we thank Drs. Michael S. Brown and Joseph L. Goldstein (University of Texas-Southwestern Medical Center, Dallas, TX) for providing us with the monoclonal antibody IgG-4A4." @default.
W2048597918 created "2016-06-24" @default.
W2048597918 creator A5061489999 @default.
W2048597918 creator A5072668138 @default.
W2048597918 date "2000-09-01" @default.
W2048597918 modified "2023-09-27" @default.
W2048597918 title "Localization of the N-terminal Domain of the Low Density Lipoprotein Receptor" @default.
W2048597918 cites W1510378561 @default.
W2048597918 cites W1554766158 @default.
W2048597918 cites W1563512755 @default.
W2048597918 cites W1622090543 @default.
W2048597918 cites W174482231 @default.
W2048597918 cites W1775749144 @default.
W2048597918 cites W1920171370 @default.
W2048597918 cites W1927237973 @default.
W2048597918 cites W1932857678 @default.
W2048597918 cites W1968343429 @default.
W2048597918 cites W1968812554 @default.
W2048597918 cites W1973979172 @default.
W2048597918 cites W1974489486 @default.
W2048597918 cites W1975771868 @default.
W2048597918 cites W1977508225 @default.
W2048597918 cites W1980092242 @default.
W2048597918 cites W1984034111 @default.
W2048597918 cites W1989293691 @default.
W2048597918 cites W1995032745 @default.
W2048597918 cites W1995077831 @default.
W2048597918 cites W2000209464 @default.
W2048597918 cites W2001337457 @default.
W2048597918 cites W2010352466 @default.
W2048597918 cites W2019708183 @default.
W2048597918 cites W2033994918 @default.
W2048597918 cites W2038138897 @default.
W2048597918 cites W2046112032 @default.
W2048597918 cites W2049556830 @default.
W2048597918 cites W2059813073 @default.
W2048597918 cites W2078508732 @default.
W2048597918 cites W2090814224 @default.
W2048597918 cites W2100837269 @default.
W2048597918 cites W2117597899 @default.
W2048597918 cites W2123259277 @default.
W2048597918 cites W2135503869 @default.
W2048597918 cites W2138466097 @default.
W2048597918 cites W2154068293 @default.
W2048597918 cites W2168656510 @default.
W2048597918 cites W2411404145 @default.
W2048597918 cites W4244423772 @default.
W2048597918 doi "https://doi.org/10.1074/jbc.m002582200" @default.
W2048597918 hasPubMedId "https://pubmed.ncbi.nlm.nih.gov/10889195" @default.
W2048597918 hasPublicationYear "2000" @default.
W2048597918 type Work @default.
W2048597918 sameAs 2048597918 @default.
W2048597918 citedByCount "10" @default.
W2048597918 countsByYear W20485979182015 @default.
W2048597918 countsByYear W20485979182016 @default.
W2048597918 crossrefType "journal-article" @default.
W2048597918 hasAuthorship W2048597918A5061489999 @default.
W2048597918 hasAuthorship W2048597918A5072668138 @default.
W2048597918 hasBestOaLocation W20485979181 @default.
W2048597918 hasConcept C12554922 @default.
W2048597918 hasConcept C134306372 @default.
W2048597918 hasConcept C170493617 @default.
W2048597918 hasConcept C174782155 @default.
W2048597918 hasConcept C185592680 @default.
W2048597918 hasConcept C2778163477 @default.
W2048597918 hasConcept C2779620165 @default.
W2048597918 hasConcept C2779664074 @default.
W2048597918 hasConcept C2780072125 @default.
W2048597918 hasConcept C33923547 @default.
W2048597918 hasConcept C36503486 @default.
W2048597918 hasConcept C41008148 @default.
W2048597918 hasConcept C43554185 @default.
W2048597918 hasConcept C55493867 @default.
W2048597918 hasConcept C76155785 @default.
W2048597918 hasConcept C8243546 @default.
W2048597918 hasConcept C86803240 @default.
W2048597918 hasConcept C95444343 @default.
W2048597918 hasConceptScore W2048597918C12554922 @default.
W2048597918 hasConceptScore W2048597918C134306372 @default.
W2048597918 hasConceptScore W2048597918C170493617 @default.
W2048597918 hasConceptScore W2048597918C174782155 @default.
W2048597918 hasConceptScore W2048597918C185592680 @default.
W2048597918 hasConceptScore W2048597918C2778163477 @default.
W2048597918 hasConceptScore W2048597918C2779620165 @default.
W2048597918 hasConceptScore W2048597918C2779664074 @default.
W2048597918 hasConceptScore W2048597918C2780072125 @default.
W2048597918 hasConceptScore W2048597918C33923547 @default.
W2048597918 hasConceptScore W2048597918C36503486 @default.
W2048597918 hasConceptScore W2048597918C41008148 @default.
W2048597918 hasConceptScore W2048597918C43554185 @default.
W2048597918 hasConceptScore W2048597918C55493867 @default.
W2048597918 hasConceptScore W2048597918C76155785 @default.
W2048597918 hasConceptScore W2048597918C8243546 @default.
W2048597918 hasConceptScore W2048597918C86803240 @default.
W2048597918 hasConceptScore W2048597918C95444343 @default.
W2048597918 hasIssue "39" @default.
W2048597918 hasLocation W20485979181 @default.
W2048597918 hasOpenAccess W2048597918 @default.