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.
Boensch
C
C
Department of Biochemistry and Molecular Biology, St. Louis University School of Medicine, Missouri 63104.
Kuo
M D
MD
Connolly
D T
DT
Huang
S S
SS
Huang
J S
JS
D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites; D001667:Binding, Competitive; D006528:Carcinoma, Hepatocellular; D002417:Cattle; D002460:Cell Line; D002462:Cell Membrane / Q000378:metabolism*; D002846:Chromatography, Affinity; D016207:Cytokines / Q000737:chemistry; D004591:Electrophoresis, Polyacrylamide Gel; D016228:Endothelial Growth Factors / Q000737:chemistry / Q000378:metabolism; D006801:Humans; D007700:Kinetics; D008099:Liver / Q000378:metabolism*; D008113:Liver Neoplasms; D008222:Lymphokines / Q000737:chemistry / Q000378:metabolism; D008565:Membrane Proteins / Q000302:isolation & purification* / Q000378:metabolism*; D008969:Molecular Sequence Data; D008970:Molecular Weight; D020575:Oncogene Proteins v-sis; D010455:Peptides / Q000138:chemical synthesis / Q000494:pharmacology; D010982:Platelet-Derived Growth Factor / Q000737:chemistry / Q000378:metabolism; D011485:Protein Binding; D011518:Proto-Oncogene Proteins / Q000737:chemistry / Q000378:metabolism; D020574:Proto-Oncogene Proteins c-sis; D015682:Retroviridae Proteins, Oncogenic / Q000737:chemistry / Q000378:metabolism; D017386:Sequence Homology, Amino Acid; D014407:Tumor Cells, Cultured; D042461:Vascular Endothelial Growth Factor A; D042442:Vascular Endothelial Growth Factors
D016207:Cytokines; D016228:Endothelial Growth Factors; D008222:Lymphokines; D008565:Membrane Proteins; D020575:Oncogene Proteins v-sis; D010455:Peptides; D010982:Platelet-Derived Growth Factor; D011518:Proto-Oncogene Proteins; D020574:Proto-Oncogene Proteins c-sis; D015682:Retroviridae Proteins, Oncogenic; D042461:Vascular Endothelial Growth Factor A; D042442:Vascular Endothelial Growth Factors; C091632:cell-surface retention-binding protein 1