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. Th is novel form of secretion appears to be mediated by cell-surface rete ntion sequences (CRS) in the polypeptide molecules. To test the hypoth esis that high-affinity CRS-binding proteins (CRS-BPs) are responsible for the cell-surface re retention, we identified and characterized th e high-affinity binding sites on various cell types for I-125-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 va scular endothelial cell growth factor, respectively. Scatchard plot an alysis revealed a single class of high-affinity binding sites with K-d = 0.5-0.7 nM and similar to 22,000-55,000 sites/cell. High-affinity b inding 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 activi ty was inhibited by heparin, polylysine, and protamine, but not by cyt ochrome c. CRS-BPs responsible for the high affinity binding were iden tified 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. Pur ified CRS-BPs exhibited ligand binding properties (pH profile and inhi bitor sensitivity) similar to those of the high-affinity binding sites for CRS pep tides on cultured cells, The major CRS BPs (p60, p66, and p72) purified from bovine liver plasma membranes were found to have i dentical N-terminal amino acid sequence and were assumed to represent different forms of the same gene product, which we have designated CRS -BP1.