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 I-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 K = 0.5-0.7 nM and 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.

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