Identification, purification, and characterization of cell-surface retention sequence-binding proteins from human SK-Hep cells and bovine liver plasma membranes.
Cell-surface retention is a newly identified mechanism associated with the secretion of certain polypeptide growth factors and cytokines. This novel form of secretion appears to be mediated by cell-surface retention sequences (CRS) in the polypeptide molecules. To test the hypothesis that high-affinity CRS-binding proteins (CRS-BPs) are responsible for the cell-surface ... retention, we identified and characterized the high-affinity binding sites on various cell types for 125I-labeled CRS peptide (sis) and CRS peptide (VEGF), each of which contained the putative CRS motifs of platelet-derived growth factor B (c-sis) and vascular endothelial cell growth factor, respectively. Scatchard plot analysis revealed a single class of high-affinity binding sites with Kd = 0.5-0.7 nM and approximately 22,000-55,000 sites/cell. High-affinity binding activity could be demonstrated between pH 4.5 and 8.0, but was much greater below 6.0 (maximum pH 5.0-5.5). The ligand binding activity was inhibited by heparin, polylysine, and protamine, but not by cytochrome c. CRS-BPs responsible for the high-affinity binding were identified as 60-72-kDa proteins by ligand affinity labeling. CRS-BPs were purified from human SK-Hep cells and bovine liver plasma membranes by Triton X-100 extraction followed by affinity column chromatography on wheat germ lectin-Sepharose 4B and CRS peptide (sis)-Affi-Gel 10. Purified CRS-BPs exhibited ligand binding properties (pH profile and inhibitor sensitivity) similar to those of the high-affinity binding sites for CRS peptides on cultured cells. The major CRS-BPs (p60, p66, and p72) purified from bovine liver plasma membranes were found to have identical N-terminal amino acid sequence and were assumed to represent different forms of the same gene product, which we have designated CRS-BP1.
Mesh Terms:
Amino Acid Sequence, Animals, Binding Sites, Binding, Competitive, Carcinoma, Hepatocellular, Cattle, Cell Line, Cell Membrane, Chromatography, Affinity, Cytokines, Electrophoresis, Polyacrylamide Gel, Endothelial Growth Factors, Humans, Kinetics, Liver, Liver Neoplasms, Lymphokines, Membrane Proteins, Molecular Sequence Data, Molecular Weight, Oncogene Proteins v-sis, Peptides, Platelet-Derived Growth Factor, Protein Binding, Proto-Oncogene Proteins, Proto-Oncogene Proteins c-sis, Retroviridae Proteins, Oncogenic, Sequence Homology, Amino Acid, Tumor Cells, Cultured, Vascular Endothelial Growth Factor A, Vascular Endothelial Growth Factors
Amino Acid Sequence, Animals, Binding Sites, Binding, Competitive, Carcinoma, Hepatocellular, Cattle, Cell Line, Cell Membrane, Chromatography, Affinity, Cytokines, Electrophoresis, Polyacrylamide Gel, Endothelial Growth Factors, Humans, Kinetics, Liver, Liver Neoplasms, Lymphokines, Membrane Proteins, Molecular Sequence Data, Molecular Weight, Oncogene Proteins v-sis, Peptides, Platelet-Derived Growth Factor, Protein Binding, Proto-Oncogene Proteins, Proto-Oncogene Proteins c-sis, Retroviridae Proteins, Oncogenic, Sequence Homology, Amino Acid, Tumor Cells, Cultured, Vascular Endothelial Growth Factor A, Vascular Endothelial Growth Factors
J. Biol. Chem.
Date: Jan. 27, 1995
PubMed ID: 7829517
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